Canine parvovirus (CPV) virus-like particle (VLP) vaccines and uses thereof

ABSTRACT

The present invention encompasses canine parvovirus (CPV) vaccines or compositions. The vaccine or composition may be a vaccine or composition containing CPV virus-like particle (VLP), and a preparation method and a use thereof. The CPV VLPs provided by the invention are formed by the CPV VP2 protein. Further, the invention broadly encompasses vaccines comprising combinations of MLV and VLP, which are capable of overcoming MDA against a variety of pathogens, which infect a variety of different species.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 62/234,196, filed on29 Sep. 2015, and herein incorporated by reference in its entirety. Allother references cited herein are similar incorporated by reference intheir entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is MER 14-252P_ST25.txt. The text file is 82 KB; itwas created on Jul. 13, 2015; and it is being submitted electronicallyvia EFS-Web, concurrent with the filing of the specification.

FIELD OF THE INVENTION

The present invention relates to the field of vaccinology, and moreparticularly, to the challenge of overcoming maternally-derivedantibodies (MDA). More particularly, the invention relates to overcomingMDA by administering to animals, including dogs, a combination ofvirus-like particles and modified live virus (MLV), either insimultaneous combination, sequential administration, or via aprime-boost administration regime. Even more particularly, the inventionrelates to compositions for eliciting protective immunity againstparvovirus (CPV) in dogs and puppies, whether or not CPV MDA are presentin the dogs and puppies.

BACKGROUND OF THE INVENTION

Canine parvovirus (CPV) is primarily an enteric pathogen that infectsdogs, especially young dogs. Parvovirus infection is characterized byacute diarrhea, fever and leukopenia in dogs and puppies more than 4 to5 weeks old, and in rare cases myocardial disease in younger puppies.The mortality rate from the disease in unvaccinated dogs is very high.And while several CPV vaccines exist, the presence of maternally-derivedantibodies (MDA) tends to block the ability of otherwise effectivevaccines to provide protective immunity.

Newborn puppies acquire passive immunities against diseases such as CPVinfection by nursing from their mother, especially during the first twodays of life. A puppy that nurses takes in colostrum in the milk that isfirst produced and (MDA) in the colostrum are passed to the puppy. Fordogs—and many other mammals—the level of passive immunity provided bythe colostrum gradually decreases as MDA are catabolized. As such, theage at which a puppy is no longer protected by MDA varies widely,depending upon the puppy's intake of colostrum, the amount of antibodiescontained therein, and several other factors.

A particular challenge when vaccinating puppies is to administervaccines according to a time frame that provides protection whichoverlaps the protection provided by maternal antibodies and begins asmaternal antibodies wane. Currently, vaccine regimens for puppiestypically begin at about 6 weeks of age and boosters are given aboutevery 3 weeks thereafter, e.g. at 9, 12 and sometimes 15 weeks. However,in order for this regimen to provide full protection, the first vaccinedose would have to immediately elicit a protective immune response. Thisexpectation is entirely unrealistic due, in part, to the immaturity ofthe puppy's immune system and the time period required to mount animmune response. Moreover, the situation is further complicated becauseresidual MDA, which may persist up to about six weeks of age, neutralizeMLV vaccines. Currently, all commercially available CPV vaccines are MLVvaccines.

Importantly, while a puppy with CPV MDA may not respond to any MLV CPVvaccine, it can still be infected by a virulent field strain of CPV, anddevelop canine parvovirosis. Because of the MDA interference, fullprotection usually does not develop until the entire course ofvaccinations is given. As a consequence, the age-based mortality due toCPV infection peaks prior to completion of vaccination protocols.Accordingly, developing a vaccine that actively immunizes puppies afterthe first injection—and in the presence of MDA—is one of the mostimportant unmet needs in canine medicine.

Another challenge in veterinary medicine is the treatment of cancer,e.g., in dogs. There are many limitations in the existing tools forcancer therapy, especially for geriatric dogs. The administration ofoncolytic parvoviruses to kill cancer cells shows great promise as aneffective cancer treatment (Rommelaere et al, Cytokine & Growth FactorReviews 21:185-195, 2010; and U.S. Pat. No. 7,179,456 to Rommelaere etal, the complete contents of which are herein incorporated by reference)and might be applied to canines. However, the existence of pre-existingantibodies to parvoviruses (e.g. as a result of vaccination) wouldrender this method ineffective, since the parvovirus would beneutralized by the existing antibodies. In addition, gene therapy indogs is rarely undertaken at present but would be a promising method fortreating several disorders, if suitable nucleic acid vectors areidentified. Accordingly, methods to overcome existing antibodies wouldbe useful for applications beyond vaccination.

In light of the above, there is a need for vaccines with an improvedsafety and a good efficacy, including the ability to overcome MDAincluding vaccines that provide protection against heterologous CPVstrains.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprisingCPV virus-like particle (VLP) antigens, CPV modified live virus (MLV)vaccines, methods of vaccination against CPV, and kits for use with suchmethods and compositions.

Compositions or vaccines comprising an antigenic CPV polypeptide andfragments and variants thereof are provided. The CPV antigens andfragments and variants thereof possess immunogenic and protectiveproperties. The CPV antigens may be produced by a baculovirus expressionvector in insect cells, and assemble into CPV empty capsids or CPV VLPs(virus-like particles).

The antigenic polypeptides and fragments and variants thereof can beformulated into vaccines with or without CPV modified live virusesand/or pharmaceutical compositions. Such vaccines or compositions can beused to vaccinate an animal and provide protection against homologousand heterologous CPV strains.

Importantly, the inventors have surprisingly and unexpectedly found thatadministration of compositions comprising both a MLV and a VLP (each anantigen corresponding to the same pathogen, but not necessarily encodingor providing the same portion or gene or subunit thereof), is capable ofovercoming MDA to elicit protective immunity against subsequent virulentchallenge by said pathogen. Accordingly, in a particular embodiment, theinvention provides combination vaccines comprising both MLV CPV and CPVVLP, which elicit protective immunity in puppies whether or not thepuppies have circulating MDA against CPV.

In another embodiment, the invention provides combination vaccinescomprising both MLV and VLP corresponding to other pathogens, where MDAinterference is a concern. For example, young bovines, porcines,felines, caprines, ovines, equines and others have circulating MDAagainst various pathogens. In each case, the presence of these MDA mayinterfere with the efficacy of vaccines. Now that this disclosure hasbeen made, the inventors envision that administering a combination ofboth MLV+VLP will overcome MDA irrespective of the pathogen. Pathogensmay include, but are not limited to: foot-and-mouth-disease virus(FMDV), porcine reproductive and respiratory syndrome virus (PRRSV),canine distemper virus (CDV), feline panleukopenia FPL, and equineinfluenza virus (EIV). The skilled person will appreciate that thisapproach, providing MLV+VLP may be applied in any case whereinterference by MDA is a challenge.

Kits comprising at least one antigenic polypeptide or fragment orvariant thereof and instructions for use are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 depicts a table summarizing the DNA and Protein sequences;

FIG. 2 depicts the plasmid map of pMEB072;

FIG. 3 depicts the plasmid map of pMEB073;

FIG. 4 is an electron micrograph of CPV VLPs, showing the correct shapeand morphology for parvovirus-like virions;

FIG. 5 is a Western Blot showing levels of CPV VLPs and accompanyingdata;

FIG. 6 is a Western Blot showing levels of CPV VLPs and accompanyingdata;

FIG. 7 is a graph showing ELISA-determined CPV titers post vaccination;

FIG. 8 is a graph showing ELISA-determined CPV titers post vaccinationwith Souriou & Bari strains (low dose, no adjuvant) or pMEB072-producedCPV VLP. Onset is more rapid in group receiving VLP;

FIG. 9 is a graph showing ELISA-determined CPV titers post vaccinationwith Souriou & Bari strains (intermediate dose, with adjuvant) orpMEB073-produced CPV VLP (with Al(OH)₃+saponin). Onset is still morerapid in group receiving VLP;

FIG. 10 is a graph showing ELISA-determined CPV titers post vaccinationwith Souriou & Bari strains (high dose) or pMEB073-produced CPV VLP(with Al(OH)₃+saponin). Onset is rapid with all three groups;

FIG. 11 is a ClustalW alignment of SEQ ID NOs: 1, 3, 4 and 6;

FIG. 12 is a graph showing individual anti-CPV antibody titers (IHA)according to treatment (MLV or MLV+VLP on D0) and day. Each linecorresponds to one individual, and the size of the points corresponds tonumber of values at the indicated combination of day and titer. Onlydata from dogs having D0 IHA titer≥40 are shown;

FIG. 13 is a graph showing mean anti-CPV antibody titers (IHA) accordingto treatment (MLV or MLV+VLP on D0) and day. Boxes represent median,25th and 75th percentiles, and ends of the whiskers represent valueswithin 1.5 times the interquartile range; each symbol represents onevalue;

FIG. 14 is a graph showing mean anti-CPV antibody titers (determined byELISA log 10 OD50) according to day post vaccination and group (Gr A:VLP SC 500 μl; Gr D VLP oral; Gr E Ad5 CPV 8.64 log 10; Gr G Ad5 CDV).Dogs were vaccinated on D0 and D28. Means are represented +/− onestandard error of the mean; each dot represents one value;

FIG. 15 is a graph showing percent responders for the study disclosed inExample 7;

FIG. 16 is a graph showing HAI titers;

FIG. 17 is a graph showing Geometric Mean Titer of CPV-neutralizingAntibodies

FIG. 18 is a graph showing Percentage Responders by Group and Day;

FIG. 19 is a graph showing CPV Geometric Mean Antibody titer by Groupand Day.

DETAILED DESCRIPTION

Compositions comprising a CPV polypeptide, antigen and fragments andvariants thereof that elicit an immunogenic response in an animal areprovided. The antigenic polypeptides or fragments or variants thereofare produced by a baculovirus expression vector in insect cells. Theantigenic polypeptides or fragments or variants may be formulated intovaccines with or without CPV modified live viruses or pharmaceuticalcompositions and used to elicit or stimulate a protective response in ananimal. In one embodiment the polypeptide antigen is a CPV capsidpolypeptide or active fragment or variant thereof. The CPV antigens maybe assembled into CPV empty capsids or CPV VLPs (virus-like particles).

It is recognized that the antigenic polypeptides of the invention may befull length polypeptides or active fragments or variants thereof. By“active fragments” or “active variants” is intended that the fragmentsor variants retain the antigenic nature of the polypeptide. Thus, thepresent invention encompasses any CPV polypeptide, antigen, epitope orimmunogen that elicits an immunogenic response in an animal. The CPVpolypeptide, antigen, epitope or immunogen may be any CPV polypeptide,antigen, epitope or immunogen, such as, but not limited to, a protein,peptide or fragment or variant thereof, that elicits, induces orstimulates a response in an animal, such as an ovine, bovine, caprine orporcine.

The present invention relates to canine vaccines or compositions whichmay comprise an effective amount of a recombinant CPV antigen. In someembodiments, the vaccines or compositions are non-adjuvanted, and maycomprise a pharmaceutically or veterinarily acceptable carrier,excipient, or vehicle.

In some embodiments, the response in the animal is a protective immuneresponse.

By “animal” it is intended mammals, birds, and the like. Animal or hostincludes mammals and human. The animal may be selected from the groupconsisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes,coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wildcats, other big cats, and other felines including cheetahs and lynx),ovine (e.g., sheep), bovine (e.g., cattle), swine (e.g., pig), caprine(e.g., goat), avian (e.g., chicken, duck, goose, turkey, quail,pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary),primate (e.g., prosimian, tarsier, monkey, gibbon, ape), and fish. Theterm “animal” also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicate otherwise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

The antigenic polypeptides of the invention are capable of protectingagainst CPV. That is, they are capable of stimulating an immune responsein an animal. By “antigen” or “immunogen” means a substance that inducesa specific immune response in a host animal. The antigen may comprise awhole organism, killed, attenuated or live; a subunit or portion of anorganism; a recombinant vector containing an insert with immunogenicproperties; a piece or fragment of DNA capable of inducing an immuneresponse upon presentation to a host animal; a polypeptide, an epitope,a hapten, or any combination thereof. Alternately, the immunogen orantigen may comprise a toxin or antitoxin.

The term “immunogenic protein, polypeptide, or peptide” as used hereinincludes polypeptides that are immunologically active in the sense thatonce administered to the host, it is able to evoke an immune response ofthe humoral and/or cellular type directed against the protein.Preferably the protein fragment is such that it has substantially thesame immunological activity as the total protein. Thus, a proteinfragment according to the invention comprises or consists essentially ofor consists of at least one epitope or antigenic determinant. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the protein, analogs thereof, or immunogenicfragments thereof. By “immunogenic fragment” is meant a fragment of aprotein which includes one or more epitopes and thus elicits theimmunological response described above. Such fragments can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes maybe determined by e.g., concurrently synthesizing large numbers ofpeptides on solid supports, the peptides corresponding to portions ofthe protein molecule, and reacting the peptides with antibodies whilethe peptides are still attached to the supports. Such techniques areknown in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysenet al., 1984; Geysen et al., 1986. Similarly, conformational epitopesare readily identified by determining spatial conformation of aminoacids such as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methodsespecially applicable to the proteins of T. parva are fully described inPCT/US2004/022605 incorporated herein by reference in its entirety.

As discussed the invention encompasses active fragments and variants ofthe antigenic polypeptide. Thus, the term “immunogenic protein,polypeptide, or peptide” further contemplates deletions, additions andsubstitutions to the sequence, so long as the polypeptide functions toproduce an immunological response as defined herein. The term“conservative variation” denotes the replacement of an amino acidresidue by another biologically similar residue, or the replacement of anucleotide in a nucleic acid sequence such that the encoded amino acidresidue does not change or is another biologically similar residue. Inthis regard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, or the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid that will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule but possessing minor amino acid substitutionsthat do not substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide. All ofthe polypeptides produced by these modifications are included herein.The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host, a quicker recovery time and/or a loweredviral titer in the infected host.

Synthetic antigens are also included within the definition, for example,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens. See, e.g., Bergmann et al., 1993; Bergmann et al.,1996; Suhrbier, 1997; Gardner et al., 1998. Immunogenic fragments, forpurposes of the present invention, will usually include at least about 3amino acids, at least about 5 amino acids, at least about 10-15 aminoacids, or about 15-25 amino acids or more amino acids, of the molecule.There is no critical upper limit to the length of the fragment, whichcould comprise nearly the full-length of the protein sequence, or even afusion protein comprising at least one epitope of the protein.

Accordingly, a minimum structure of a polynucleotide expressing anepitope is that it comprises or consists essentially of or consists ofnucleotides encoding an epitope or antigenic determinant of a CPVpolypeptide. A polynucleotide encoding a fragment of a CPV polypeptidemay comprise or consist essentially of or consist of a minimum of 15nucleotides, about 30-45 nucleotides, about 45-75, or at least 57, 87 or150 consecutive or contiguous nucleotides of the sequence encoding thepolypeptide. Epitope determination procedures, such as, generatingoverlapping peptide libraries (Hemmer et al., 1998), Pepscan (Geysen etal., 1984; Geysen et al., 1985; Van der Zee R. et al., 1989; Geysen,1990; Multipin® Peptide Synthesis Kits de Chiron) and algorithms (DeGroot et al., 1999; PCT/US2004/022605) can be used in the practice ofthe invention.

The term “nucleic acid” and “polynucleotide” refers to RNA or DNA thatis linear or branched, single or double stranded, or a hybrid thereof.The term also encompasses RNA/DNA hybrids. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs, uracyl, other sugars andlinking groups such as fluororibose and thiolate, and nucleotidebranches. The sequence of nucleotides may be further modified afterpolymerization, such as by conjugation, with a labeling component. Othertypes of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides orsolid support. The polynucleotides can be obtained by chemical synthesisor derived from a microorganism.

The term “gene” is used broadly to refer to any segment ofpolynucleotide associated with a biological function. Thus, genesinclude introns and exons as in genomic sequence, or just the codingsequences as in cDNAs and/or the regulatory sequences required for theirexpression. For example, gene also refers to a nucleic acid fragmentthat expresses mRNA or functional RNA, or encodes a specific protein,and which includes regulatory sequences.

The invention further comprises a complementary strand to apolynucleotide encoding a CPV antigen, epitope or immunogen. Thecomplementary strand can be polymeric and of any length, and can containdeoxyribonucleotides, ribonucleotides, and analogs in any combination.

The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer can be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

An “isolated” biological component (such as a nucleic acid or protein ororganelle) refers to a component that has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinanttechnology as well as chemical synthesis.

The term “purified” as used herein does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedpolypeptide preparation is one in which the polypeptide is more enrichedthan the polypeptide is in its natural environment. That is thepolypeptide is separated from cellular components. By “substantiallypurified” it is intended that such that the polypeptide representsseveral embodiments at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or at least 98%, or more of the cellular componentsor materials have been removed. Likewise, the polypeptide may bepartially purified. By “partially purified” is intended that less than60% of the cellular components or material is removed. The same appliesto polynucleotides. The polypeptides disclosed herein can be purified byany of the means known in the art.

As noted above, the antigenic polypeptides or fragments or variantsthereof are CPV antigenic polypeptides that are produced by abaculovirus expression vector in insect cells. Fragments and variants ofthe disclosed polynucleotides and polypeptides encoded thereby are alsoencompassed by the present invention. By “fragment” is intended aportion of the polynucleotide or a portion of the antigenic amino acidsequence encoded thereby. Fragments of a polynucleotide may encodeprotein fragments that retain the biological activity of the nativeprotein and hence have immunogenic activity as noted elsewhere herein.Fragments of the polypeptide sequence retain the ability to induce aprotective immune response in an animal.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. Variants of a particularpolynucleotide of the invention (i.e., the reference polynucleotide) canalso be evaluated by comparison of the percent sequence identity betweenthe polypeptide encoded by a variant polynucleotide and the polypeptideencoded by the reference polynucleotide. “Variant” protein is intendedto mean a protein derived from the native protein by deletion oraddition of one or more amino acids at one or more sites in the nativeprotein and/or substitution of one or more amino acids at one or moresites in the native protein. Variant proteins encompassed by the presentinvention are biologically active, that is they the ability to elicit animmune response.

In one aspect, the present invention provides CPV polypeptides from CPVisolates. In another aspect, the present invention provides apolypeptide having a sequence as set forth in SEQ ID NOs: 1, 3, 4, 6,8-10, and variant or fragment thereof.

In another aspect, the invention relates to CPV empty capsids or CPVVLPs (virus-like particles). The capsids may comprise, consistessentially of, or consist of CPV VP2 polypeptides, or variants,including truncated versions thereof.

Moreover, homologs of CPV polypeptides are intended to be within thescope of the present invention. As used herein, the term “homologs”includes orthologs, analogs and paralogs. The term “analogs” refers totwo polynucleotides or polypeptides that have the same or similarfunction, but that have evolved separately in unrelated organisms. Theterm “orthologs” refers to two polynucleotides or polypeptides fromdifferent species, but that have evolved from a common ancestral gene byspeciation. Normally, orthologs encode polypeptides having the same orsimilar functions. The term “paralogs” refers to two polynucleotides orpolypeptides that are related by duplication within a genome. Paralogsusually have different functions, but these functions may be related.Analogs, orthologs, and paralogs of a wild-type CPV polypeptide candiffer from the wild-type CPV polypeptide by post-translationalmodifications, by amino acid sequence differences, or by both. Inparticular, homologs of the invention will generally exhibit at least80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, 99% sequence identity,with all or part of the wild-type CPV polynucleotide sequences, and willexhibit a similar function. Variants include allelic variants. The term“allelic variant” refers to a polynucleotide or a polypeptide containingpolymorphisms that lead to changes in the amino acid sequences of aprotein and that exist within a natural population (e.g., a virusspecies or variety). Such natural allelic variations can typicallyresult in 1-5% variance in a polynucleotide or a polypeptide. Allelicvariants can be identified by sequencing the nucleic acid sequence ofinterest in a number of different species, which can be readily carriedout by using hybridization probes to identify the same gene geneticlocus in those species. Any and all such nucleic acid variations andresulting amino acid polymorphisms or variations that are the result ofnatural allelic variation and that do not alter the functional activityof gene of interest, are intended to be within the scope of theinvention.

As used herein, the term “derivative” or “variant” refers to apolypeptide, or a nucleic acid encoding a polypeptide, that has one ormore conservative amino acid variations or other minor modificationssuch that (1) the corresponding polypeptide has substantially equivalentfunction when compared to the wild type polypeptide or (2) an antibodyraised against the polypeptide is immunoreactive with the wild-typepolypeptide. These variants or derivatives include polypeptides havingminor modifications of the CPV polypeptide primary amino acid sequencesthat may result in peptides which have substantially equivalent activityas compared to the unmodified counterpart polypeptide. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. The term “variant” further contemplates deletions,additions and substitutions to the sequence, so long as the polypeptidefunctions to produce an immunological response as defined herein.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.In this regard, particularly preferred substitutions will generally beconservative in nature, as described above.

The polynucleotides of the disclosure include sequences that aredegenerate as a result of the genetic code, e.g., optimized codon usagefor a specific host. As used herein, “optimized” refers to apolynucleotide that is genetically engineered to increase its expressionin a given species. To provide optimized polynucleotides coding for CPVpolypeptides, the DNA sequence of the CPV protein gene can be modifiedto 1) comprise codons preferred by highly expressed genes in aparticular species; 2) comprise an A+T or G+C content in nucleotide basecomposition to that substantially found in said species; 3) form aninitiation sequence of said species; or 4) eliminate sequences thatcause destabilization, inappropriate polyadenylation, degradation andtermination of RNA, or that form secondary structure hairpins or RNAsplice sites. Increased expression of CPV protein in said species can beachieved by utilizing the distribution frequency of codon usage ineukaryotes and prokaryotes, or in a particular species. The term“frequency of preferred codon usage” refers to the preference exhibitedby a specific host cell in usage of nucleotide codons to specify a givenamino acid. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in the disclosure as long as the amino acidsequence of the CPV polypeptide encoded by the nucleotide sequence isfunctionally unchanged.

The sequence identity between two amino acid sequences may beestablished by the NCBI (National Center for Biotechnology Information)pairwise blast and the blosum62 matrix, using the standard parameters(see, e.g., the BLAST or BLASTX algorithm available on the “NationalCenter for Biotechnology Information” (NCBI, Bethesda, Md., USA) server,as well as in Altschul et al.; and thus, this document speaks of usingthe algorithm or the BLAST or BLASTX and BLOSUM62 matrix by the term“blasts”).

The “identity” with respect to sequences can refer to the number ofpositions with identical nucleotides or amino acids divided by thenumber of nucleotides or amino acids in the shorter of the two sequenceswherein alignment of the two sequences can be determined in accordancewith the Wilbur and Lipman algorithm (Wilbur and Lipman), for instance,using a window size of 20 nucleotides, a word length of 4 nucleotides,and a gap penalty of 4, and computer-assisted analysis andinterpretation of the sequence data including alignment can beconveniently performed using commercially available programs (e.g.,Intelligenetics™ Suite, Intelligenetics Inc. CA). When RNA sequences aresaid to be similar, or have a degree of sequence identity or homologywith DNA sequences, thymidine (T) in the DNA sequence is consideredequal to uracil (U) in the RNA sequence. Thus, RNA sequences are withinthe scope of the invention and can be derived from DNA sequences, bythymidine (T) in the DNA sequence being considered equal to uracil (U)in RNA sequences.

The sequence identity or sequence similarity of two amino acidsequences, or the sequence identity between two nucleotide sequences canbe determined using Vector NTI software package (Invitrogen, 1600Faraday Ave., Carlsbad, Calif.).

The following documents provide algorithms for comparing the relativeidentity or homology of sequences, and additionally or alternativelywith respect to the foregoing, the teachings in these references can beused for determining percent homology or identity: Needleman S B andWunsch C D; Smith T F and Waterman M S; Smith T F, Waterman M S andSadler J R; Feng D F and Dolittle R F; Higgins D G and Sharp P M;Thompson J D, Higgins D G and Gibson T J; and, Devereux J, Haeberlie Pand Smithies O. And, without undue experimentation, the skilled artisancan consult with many other programs or references for determiningpercent homology.

Hybridization reactions can be performed under conditions of different“stringency.” Conditions that increase stringency of a hybridizationreaction are well known. See for example, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989).

The invention further encompasses the CPV polynucleotides contained in avector molecule or an expression vector and operably linked to apromoter element and optionally to an enhancer.

A “vector” refers to a recombinant DNA or RNA plasmid or virus thatcomprises a heterologous polynucleotide to be delivered to a targetcell, either in vitro or in vivo. The heterologous polynucleotide maycomprise a sequence of interest for purposes of prevention or therapy,and may optionally be in the form of an expression cassette. As usedherein, a vector needs not be capable of replication in the ultimatetarget cell or subject. The term includes cloning vectors and viralvectors.

The term “recombinant” means a polynucleotide semisynthetic, orsynthetic origin which either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

“Heterologous” means derived from a genetically distinct entity from therest of the entity to which it is being compared. For example, apolynucleotide may be placed by genetic engineering techniques into aplasmid or vector derived from a different source, and is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous promoter.

The present invention relates to ovine, bovine, caprine and porcinevaccines or pharmaceutical or immunological compositions which maycomprise an effective amount of a recombinant CPV antigens and apharmaceutically or veterinarily acceptable carrier, adjuvant,excipient, or vehicle.

The subject matter described herein is directed in part, to compositionsand methods related to the CPV antigen prepared in a baculovirus/insectcell expression system that was highly immunogenic and protected animalsagainst challenge from homologous and heterologous CPV strains.

Compositions

The present invention relates to a CPV vaccine or composition which maycomprise an effective amount of a recombinant CPV antigen and apharmaceutically or veterinarily acceptable carrier, excipient, orvehicle. In one embodiment, the recombinant CPV antigen is expressed bya baculovirus expression vector in insect cells.

One embodiment of the invention relates to a vaccine or compositioncomprising CPV empty capsids or CPV VLPs (virus-like particles). The CPVempty capsids or CPV VLPs (virus-like particles) are obtained byexpression of the CPV capsid protein.

The present invention also relates to processes for preparing thesevaccines, the use of antigens for producing these vaccines andvaccination methods using them.

The present invention also relates to nucleotide sequences, inparticular cDNA, and to amino acid sequences, modified compared withnatural sequences of the virus. The invention also relates to theexpression products of the modified nucleotide sequences and to the CPVantigens and virus incorporating these modifications.

The present invention encompasses any CPV polypeptide, antigen, epitopeor immunogen that elicits an immunogenic response in an animal, such asan ovine, bovine, caprine or swine. The CPV polypeptide, antigen,epitope or immunogen may be any CPV polypeptide, antigen, epitope orimmunogen, such as, but not limited to, a protein, peptide or fragmentthereof, that elicits, induces or stimulates a response in an animal,such as canine.

In an embodiment wherein the CPV immunological composition or vaccine isa recombinant immunological composition or vaccine, the composition orvaccine comprising a recombinant vector and is non-adjuvanted, and mayoptionally comprise a pharmaceutical or veterinary acceptable excipient,carrier or vehicle; the recombinant vector is a baculovirus expressionvector which may comprise a polynucleotide encoding a CPV polypeptide,antigen, epitope or immunogen. The CPV polypeptide, antigen, epitope orimmunogen, may be capsid protein and any fragment thereof.

In one embodiment, the nucleic acid molecule encoding one or more CPVantigen(s) is a cDNA encoding a CPV capsid protein. In anotherembodiment, the nucleic acid molecule encoding one or more CPVantigen(s) is a cDNA encoding a fragment of the CPV capsid protein.

In another embodiment, the CPV antigen may be derived from CPV strain100869-1.

The present invention relates to a CPV composition or vaccine which maycomprise an effective amount of a recombinant CPV antigen. The CPVcomposition or vaccine does not contain an adjuvant. The CPV compositionor vaccine may optionally contain a pharmaceutically or veterinarilyacceptable carrier, excipient, or vehicle.

The invention further encompasses the CPV polynucleotides contained in avector molecule or an expression vector and operably linked to apromoter element and optionally to an enhancer.

In one aspect, the present invention provides CPV polypeptides having asequence as set forth in SEQ ID NO: 1, 3, 4, 6, or 8-10, and variants orfragments thereof.

In another aspect, the present invention provides a polypeptide havingat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 96%, 97%, 98% or 99% sequence identity to an antigenicpolypeptide of the invention, particularly to the polypeptides having asequence as set forth in SEQ ID NO: 1, 3, 4, 6, or 8-10.

In yet another aspect, the present invention provides fragments andvariants of the CPV polypeptides identified above (SEQ ID NO: 1, 3, 4,6, or 8-10) which may readily be prepared by one of skill in the artusing well-known molecular biology techniques.

Variants are homologous polypeptides having an amino acid sequence atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to theamino acid sequence as set forth in SEQ ID NO: 1, 3, 4, 6, or 8-10.

An immunogenic fragment of a CPV polypeptide includes at least 8, 10,15, or 20 consecutive amino acids, at least 21 amino acids, at least 23amino acids, at least 25 amino acids, or at least 30 amino acids of aCPV polypeptide having a sequence as set forth in SEQ ID NO: 1, 3, 4, 6,or 8-10, or variants thereof. In another embodiment, a fragment of a CPVpolypeptide includes a specific antigenic epitope found on a full-lengthCPV polypeptide. However, the skilled person will understand that asufficient portion of the CPV polypeptide must be present to enableformation of CPV VLPs.

In another aspect, the present invention provides a polynucleotideencoding a CPV polypeptide, such as a polynucleotide encoding apolypeptide having a sequence as set forth in SEQ ID NO: 1, 3, 4, 6, or8-10. In yet another aspect, the present invention provides apolynucleotide encoding a polypeptide having at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or99% sequence identity to a polypeptide having a sequence as set forth inSEQ ID NO: 1, 3, 4, 6, or 8-10, or a conservative variant, an allelicvariant, a homolog or an immunogenic fragment comprising at least eightor at least ten consecutive amino acids of one of these polypeptides, ora combination of these polypeptides.

In another aspect, the present invention provides a polynucleotidehaving a nucleotide sequence as set forth in SEQ ID NO: 2, 5, 7, 11 or12, or a variant thereof. In yet another aspect, the present inventionprovides a polynucleotide having at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 95%, 96%, 97%,98%, or 99% sequence identity to one of a polynucleotide having asequence as set forth in SEQ ID NO: 2, 5, 7, 11 or 12, or a variantthereof.

The polynucleotides of the invention may comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,5′UTR, 3′UTR, transcription terminators, polyadenylation sites,additional transcription units under control of the same or a differentpromoter, sequences that permit cloning, expression, homologousrecombination, and transformation of a host cell, and any such constructas may be desirable to provide embodiments of this invention.

Elements for the expression of a CPV polypeptide, antigen, epitope orimmunogen are advantageously present in an inventive vector. In minimummanner, this comprises, consists essentially of, or consists of aninitiation codon (ATG), a stop codon and a promoter, and optionally alsoa polyadenylation sequence for certain vectors such as plasmid andcertain viral vectors, e.g., viral vectors other than poxviruses. Whenthe polynucleotide encodes a polyprotein fragment, e.g. a CPV peptide,advantageously, in the vector, an ATG is placed at 5′ of the readingframe and a stop codon is placed at 3′. Other elements for controllingexpression may be present, such as enhancer sequences, stabilizingsequences, such as intron and signal sequences permitting the secretionof the protein.

The present invention also relates to preparations comprising vectors,such as expression vectors, e.g., therapeutic compositions. Thepreparations can comprise one or more vectors, e.g., expression vectors,such as in vivo expression vectors, comprising and expressing one ormore CPV polypeptides, antigens, epitopes or immunogens. In oneembodiment, the vector contains and expresses a polynucleotide thatcomprises, consists essentially of, or consists of a polynucleotidecoding for (and advantageously expressing) a CPV antigen, epitope orimmunogen, in a pharmaceutically or veterinarily acceptable carrier,excipient or vehicle. Thus, according to an embodiment of the invention,the other vector or vectors in the preparation comprises, consistsessentially of or consists of a polynucleotide that encodes, and underappropriate circumstances the vector expresses one or more otherproteins of a CPV polypeptide, antigen, epitope or immunogen, or afragment thereof.

According to another embodiment, the vector or vectors in thepreparation comprise, or consist essentially of, or consist ofpolynucleotide(s) encoding one or more proteins or fragment(s) thereofof a CPV polypeptide, antigen, epitope or immunogen, the vector orvectors expressing the polynucleotide(s). In another embodiment, thepreparation comprises one, two, or more vectors comprisingpolynucleotides encoding and expressing, advantageously in vivo, a CPVpolypeptide, antigen, fusion protein or an epitope thereof.

According to a yet further embodiment of the invention, the expressionvector is a plasmid vector or a DNA plasmid vector, in particular an invivo expression vector. In a specific, non-limiting example, the pVR1020or 1012 plasmid (VICAL Inc.; Luke et al., 1997; Hartikka et al., 1996,see, e.g., U.S. Pat. Nos. 5,846,946 and 6,451,769) can be utilized as avector for the insertion of a polynucleotide sequence. The pVR1020plasmid is derived from pVR1012 and contains the human tPA signalsequence. In one embodiment the human tPA signal comprises from aminoacid M(1) to amino acid S(23) in Genbank under the accession numberHUMTPA14. In another specific, non-limiting example, the plasmidutilized as a vector for the insertion of a polynucleotide sequence cancontain the signal peptide sequence of equine IGF1 from amino acid M(24)to amino acid A(48) in Genbank under the accession number U28070.Additional information on DNA plasmids which may be consulted oremployed in the practice are found, for example, in U.S. Pat. Nos.6,852,705; 6,818,628; 6,586,412; 6,576,243; 6,558,674; 6,464,984;6,454770; 6,376,473 and 6,221,362.

The term plasmid covers any DNA transcription unit comprising apolynucleotide according to the invention and the elements necessary forits in vivo expression in a cell or cells of the desired host or target;and, in this regard, it is noted that a supercoiled or non-supercoiled,circular plasmid, as well as a linear form, are intended to be withinthe scope of the invention.

Each plasmid comprises or contains or consists essentially of, inaddition to the polynucleotide encoding a CPV antigen, epitope orimmunogen, optionally fused with a heterologous peptide sequence,variant, analog or fragment, operably linked to a promoter or under thecontrol of a promoter or dependent upon a promoter. In general, it isadvantageous to employ a strong promoter functional in eukaryotic cells.The strong promoter may be, but not limited to, the immediate earlycytomegalovirus promoter (CMV-IE) of human or murine origin, oroptionally having another origin such as the rat or guinea pig, theSuper promoter (Ni, M. et al., Plant J. 7, 661-676, 1995.). The CMV-IEpromoter can comprise the actual promoter part, which may or may not beassociated with the enhancer part. Reference can be made to EP-A-260148, EP-A-323 597, U.S. Pat. Nos. 5,168,062, 5,385,839, and 4,968,615,as well as to PCT Application No WO87/03905. The CMV-IE promoter isadvantageously a human CMV-IE (Boshart et al., 1985) or murine CMV-IE.

In more general terms, the promoter has either a viral, a plant, or acellular origin. A strong viral promoter other than CMV-IE that may beusefully employed in the practice of the invention is the early/latepromoter of the SV40 virus or the LTR promoter of the Rous sarcomavirus. A strong cellular promoter that may be usefully employed in thepractice of the invention is the promoter of a gene of the cytoskeleton,such as e.g. the desmin promoter (Kwissa et al., 2000), or the actinpromoter (Miyazaki et al., 1989).

The plasmids may comprise other expression control elements. It isparticularly advantageous to incorporate stabilizing sequence(s), e.g.,intron sequence(s), for example, maize alcohol dehydrogenase intron(Callis et al. Genes & Dev. 1(10):1183-1200, December 1987), the firstintron of the hCMV-IE (PCT Application No. WO1989/01036), the intron IIof the rabbit β-globin gene (van Ooyen et al., 1979). In anotherembodiment, the plasmids may comprise 3′ UTR. The 3′ UTR may be, but notlimited to, agrobacterium nopaline synthase (Nos) 3′ UTR (Nopalinesynthase: transcript mapping and DNA sequence. Depicker, A. et al. J.Mol. Appl. Genet., 1982; Bevan, NAR, 1984, 12(22): 8711-8721).

As to the polyadenylation signal (polyA) for the plasmids and viralvectors other than poxviruses, use can more be made of the poly(A)signal of the bovine growth hormone (bGH) gene (see U.S. Pat. No.5,122,458), or the poly(A) signal of the rabbit β-globin gene or thepoly(A) signal of the SV40 virus.

A “host cell” denotes a prokaryotic or eukaryotic cell that has beengenetically altered, or is capable of being genetically altered byadministration of an exogenous polynucleotide, such as a recombinantplasmid or vector. When referring to genetically altered cells, the termrefers both to the originally altered cell and to the progeny thereof.

In one embodiment, the recombinant CPV antigen is expressed in insectcells.

In one particular embodiment, the CPV antigen is expressed in SF9 cells.

Methods of Use

In an embodiment, the subject matter disclosed herein is directed to amethod of vaccinating an ovine, bovine, caprine, or swine comprisingadministering to the ovine, bovine, caprine, or swine an effectiveamount of a vaccine which may comprise an effective amount of arecombinant CPV antigen and a pharmaceutically or veterinarilyacceptable carrier, excipient, or vehicle.

In one embodiment of the present invention, the method comprises asingle administration of a vaccine composition formulated with anemulsion according to the invention. For example, in one embodiment, theimmunological or vaccine composition comprises baculovirus expressed CPVantigens, including polypeptides and VLPs (virus-like particles) orempty capsids. Electron microscopy indicates the insect cellstransformed with baculovirus expression vectors produce CPV VLPs or CPVempty capsids, and so immunological or vaccine compositions according tothe instant invention encompass those comprising CPV VLPs or CPV emptycapsids.

In an embodiment, the subject matter disclosed herein is directed to amethod of vaccinating an ovine, bovine, caprine, or swine comprisingadministering to the ovine, bovine, caprine, or swine the CPV antigenproduced by a baculovirus vector in insect cells.

In an embodiment, the subject matter disclosed herein is directed to amethod of eliciting an immune response comprising administering to theovine, bovine, caprine, or swine a vaccine comprising the CPV antigenproduced by a baculovirus vector in insect cells.

In an embodiment, the subject matter disclosed herein is directed to amethod of preparing a vaccine or composition comprising isolating a CPVantigen produced by a baculovirus vector in insect cells and optionallycombining with a pharmaceutically or veterinarily acceptable carrier,excipient or vehicle.

Both homologous and heterologous CPV strains are used for challenge totest the efficacy of the vaccine. The administering may besubcutaneously or intramuscularly. The administering may be needle free(for example, Bioject).

In one embodiment of the invention, a prime-boost regimen can beemployed, which is comprised of at least one primary administration andat least one booster administration using at least one commonpolypeptide, antigen, epitope or immunogen. Typically the immunologicalcomposition or vaccine used in primary administration is different innature from those used as a booster. However, it is noted that the samecomposition can be used as the primary administration and the boost.This administration protocol is called “prime-boost”.

A prime-boost according to the present invention can include arecombinant viral vector is used to express a CPV coding sequence orfragments thereof encoding an antigenic polypeptide or fragment orvariant thereof. Specifically, the viral vector can express a CPV geneor fragment thereof that encodes an antigenic polypeptide. Viral vectorcontemplated herein includes, but not limited to, poxvirus [e.g.,vaccinia virus or attenuated vaccinia virus, avipox virus or attenuatedavipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox,ALVAC, TROVAC; see e.g., U.S. Pat. No. 5,505,941, U.S. Pat. No.5,494,8070), raccoonpox virus, swinepox virus, etc.], adenovirus (e.g.,human adenovirus, canine adenovirus), herpesvirus (e.g. canineherpesvirus, herpesvirus of turkey, Marek's disease virus, infectiouslaryngotracheitis virus, feline herpesvirus, laryngotracheitis virus(ILTV), bovine herpesvirus, swine herpesvirus), baculovirus, retrovirus,etc. In another embodiment, the avipox expression vector may be acanarypox vector, such as, ALVAC. In yet another embodiment, the avipoxexpression vector may be a fowlpox vector, such as, TROVAC. The CPVantigen of the invention to be expressed is inserted under the controlof a specific poxvirus promoter, e.g., the entomopoxvirus Amsacta moorei42K promoter (Barcena, Lorenzo et al. 2000), the vaccinia promoter 7.5kDa (Cochran et al., 1985), the vaccinia promoter I3L (Riviere et al.,1992), the vaccinia promoter HA (Shida, 1986), the cowpox promoter ATI(Funahashi et al., 1988), the vaccinia promoter H6 (Taylor et al.,1988b; Guo et al., 1989; Perkus et al., 1989), inter alia.

In another aspect of the prime-boost protocol of the invention, acomposition comprising the CPV antigen of the invention is administeredfollowed by the administration of vaccine or composition comprising arecombinant viral vector that contains and expresses the CPV antigen invivo, or an inactivated viral vaccine or composition comprising the CPVantigen, or a DNA plasmid vaccine or composition that contains orexpresses the CPV antigen. Likewise, a prime-boost protocol may comprisethe administration of vaccine or composition comprising a recombinantviral vector that contains and expresses a CPV antigen in vivo, or aninactivated viral vaccine or composition comprising a CPV antigen, or aDNA plasmid vaccine or composition that contains or expresses a CPVantigen, followed by the administration of a composition comprising theCPV antigen of the invention. It is further noted that both the primaryand the secondary administrations may comprise the compositioncomprising the CPV antigen of the invention.

A prime-boost protocol comprises at least one prime-administration andat least one boost administration using at least one common polypeptideand/or variants or fragments thereof. The vaccine used inprime-administration may be different in nature from those used as alater booster vaccine. The prime-administration may comprise one or moreadministrations. Similarly, the boost administration may comprise one ormore administrations.

The dose volume of compositions for target species that are mammalsbased on viral vectors, e.g., non-poxvirus-viral-vector-basedcompositions, is generally between about 0.1 to about 5.0 ml, betweenabout 0.1 to about 3.0 ml, and between about 0.5 ml to about 2.5 ml.

The efficacy of the vaccines may be tested about 2 to 4 weeks after thelast immunization by challenging animals, such as feline or canine, witha virulent strain of CPV.

Further details of these CPV strains may be found on the EuropeanBioinformatics Information (EMBL-EBI) web pages, and all of theassociated nucleotide sequences are herein incorporated by reference.The inventors contemplate that all CPV strains, both herein listed, andthose yet to be identified, could be expressed according to theteachings of the present disclosure to produce, for example, effectivevaccine compositions. Both homologous and heterologous strains are usedfor challenge to test the efficacy of the vaccines. The animal may bechallenged intradermally, subcutaneously, spray, intra-nasally,intra-ocularly, intra-tracheally, and/or orally.

The prime-boost administrations may be advantageously carried out 1 to 6weeks apart, for example, about 4 weeks apart. According to oneembodiment, a semi-annual booster or an annual booster, advantageouslyusing the viral vector-based vaccine, is also envisaged. The animals areadvantageously at least 6 to 8 weeks old at the time of the firstadministration.

The compositions comprising the recombinant antigenic polypeptides ofthe invention used in the prime-boost protocols are not adjuvanted, andmay optionally be contained in a pharmaceutically or veterinaryacceptable vehicle, diluent or excipient. The protocols of the inventionprotect the animal from CPV and/or prevent disease progression in aninfected animal.

It should be understood by one of skill in the art that the disclosureherein is provided by way of example and the present invention is notlimited thereto. From the disclosure herein and the knowledge in theart, the skilled artisan can determine the number of administrations,the administration route, and the doses to be used for each injectionprotocol, without any undue experimentation.

The present invention contemplates at least one administration to ananimal of an efficient amount of the therapeutic composition madeaccording to the invention. The animal may be male, female, pregnantfemale and newborn. This administration may be via various routesincluding, but not limited to, intramuscular (IM), intradermal (ID) orsubcutaneous (SC) injection or via intranasal or oral administration.The therapeutic composition according to the invention can also beadministered by a needleless apparatus (as, for example with a Pigjet,Dermojet, Biojector, Avijet (Merial, Ga., USA), Vetjet or Vitajetapparatus (Bioject, Oreg., USA)). Another approach to administeringplasmid compositions is to use electroporation (see, e.g. Tollefsen etal., 2002; Tollefsen et al., 2003; Babiuk et al., 2002; PCT ApplicationNo. WO99/01158). In another embodiment, the therapeutic composition isdelivered to the animal by gene gun or gold particle bombardment.

In one embodiment, the invention provides for the administration of atherapeutically effective amount of a formulation for the delivery andexpression of a CPV antigen or epitope in a target cell. Determinationof the therapeutically effective amount is routine experimentation forone of ordinary skill in the art. In one embodiment, the formulationcomprises an expression vector comprising a polynucleotide thatexpresses a CPV antigen or epitope and a pharmaceutically orveterinarily acceptable carrier, vehicle or excipient. In anotherembodiment, the pharmaceutically or veterinarily acceptable carrier,vehicle or excipient facilitates transfection or other means of transferof polynucleotides to a host animal and/or improves preservation of thevector or protein in a host.

In one embodiment, the subject matter disclosed herein provides adetection method for differentiation between infected and vaccinatedanimals (DIVA).

It is disclosed herein that the use of the vaccine or composition of thepresent invention allows the detection of CPV infection in an animal. Itis disclosed herein that the use of the vaccine or composition of thepresent invention allows the detection of the infection in animals bydifferentiating between infected and vaccinated animals (DIVA).

Article of Manufacture

In an embodiment, the subject matter disclosed herein is directed to akit for performing a method of eliciting or inducing an immune responsewhich may comprise any one of the recombinant CPV immunologicalcompositions or vaccines, or inactivated CPV immunological compositionsor vaccines, recombinant CPV viral compositions or vaccines, andinstructions for performing the method.

Another embodiment of the invention is a kit for performing a method ofinducing an immunological or protective response against CPV in ananimal comprising a composition or vaccine comprising a CPV antigen ofthe invention and a recombinant CPV viral immunological composition orvaccine, and instructions for performing the method of delivery in aneffective amount for eliciting an immune response in the animal.

Another embodiment of the invention is a kit for performing a method ofinducing an immunological or protective response against CPV in ananimal comprising a composition or vaccine comprising a CPV antigen ofthe invention and an inactivated CPV immunological composition orvaccine, and instructions for performing the method of delivery in aneffective amount for eliciting an immune response in the animal.

Yet another aspect of the present invention relates to a kit forprime-boost vaccination according to the present invention as describedabove. The kit may comprise at least two vials: a first vial containinga vaccine or composition for the prime-vaccination according to thepresent invention, and a second vial containing a vaccine or compositionfor the boost-vaccination according to the present invention. The kitmay advantageously contain additional first or second vials foradditional primo-vaccinations or additional boost-vaccinations.

The following embodiments are encompassed by the invention. In anembodiment, a composition comprising a CPV antigen or fragment orvariant thereof and a pharmaceutical or veterinarily acceptable carrier,excipient, or vehicle is disclosed. In another embodiment, thecomposition described above wherein the CPV antigen or fragment orvariant thereof comprises an immunogenic fragment comprising at least 15amino acids of a CPV antigen is disclosed. In an embodiment, the abovecompositions wherein the CPV antigen or fragment or variant thereof ispartially purified are disclosed. In an embodiment, the abovecompositions wherein the CPV antigen or fragment or variant thereof issubstantially purified are disclosed.

In an embodiment, the above compositions wherein the CPV antigen orfragment or variant thereof is a CPV polypeptide are disclosed. In anembodiment, the above compositions wherein the CPV polypeptide is acapsid protein or a fragment thereof are disclosed. In an embodiment,the above compositions wherein the CPV antigen or fragment or variantthereof has at least 80% sequence identity to the sequence as set forthin SEQ ID NO: 1, 3, 4, 6, or 8-10 are disclosed. In one embodiment, theabove compositions wherein the CPV antigen is encoded by apolynucleotide having at least 70% sequence identity to the sequence asset forth in SEQ ID NO: 2, 5, 7, 11 or 12 are disclosed. In anotherembodiment, a method of vaccinating an animal susceptible to CPVcomprising administering the compositions above to the animal isdisclosed. In an embodiment, a method of vaccinating an animalsusceptible to CPV comprising a prime-boost regime is disclosed. In anembodiment, a substantially purified antigenic polypeptide expressed ininsect cells, wherein the polypeptide comprises: an amino acid sequencehaving at least 80% sequence identity to a polypeptide having thesequence as set forth in SEQ ID NO: 1, 3, 4, 6, or 8-10 is disclosed. Inany embodiment the animal is preferably a feline or canine. In oneembodiment, a method of diagnosing CPV infection in an animal isdisclosed. In yet another embodiment, a kit for prime-boost vaccinationcomprising at least two vials, wherein a first vial containing thecomposition of the present invention, and a second vial containing acomposition for the boost-vaccination comprising a compositioncomprising a recombinant viral vector, or a composition comprising aninactivated viral composition, or a DNA plasmid composition thatcontains or expresses the CPV antigen is disclosed.

The pharmaceutically or veterinarily acceptable carriers or vehicles orexcipients are well known to the one skilled in the art. For example, apharmaceutically or veterinarily acceptable carrier or vehicle orexcipient can be a 0.9% NaCl (e.g., saline) solution or a phosphatebuffer. Other pharmaceutically or veterinarily acceptable carrier orvehicle or excipients that can be used for methods of this inventioninclude, but are not limited to, poly-(L-glutamate) orpolyvinylpyrrolidone. The pharmaceutically or veterinarily acceptablecarrier or vehicle or excipients may be any compound or combination ofcompounds facilitating the administration of the vector (or proteinexpressed from an inventive vector in vitro); advantageously, thecarrier, vehicle or excipient may facilitate transfection and/or improvepreservation of the vector (or protein). Doses and dose volumes areherein discussed in the general description and can also be determinedby the skilled artisan from this disclosure read in conjunction with theknowledge in the art, without any undue experimentation.

The cationic lipids containing a quaternary ammonium salt which areadvantageously but not exclusively suitable for plasmids, areadvantageously those having the following formula:

in which R1 is a saturated or unsaturated straight-chain aliphaticradical having 12 to 18 carbon atoms, R2 is another aliphatic radicalcontaining 2 or 3 carbon atoms and X is an amine or hydroxyl group, e.g.the DMRIE. In another embodiment the cationic lipid can be associatedwith a neutral lipid, e.g. the DOPE.

Among these cationic lipids, preference is given to DMRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium; WO96/34109), advantageously associated with a neutral lipid,advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr, 1994),to form DMRIE-DOPE.

Advantageously, the plasmid mixture with the adjuvant is formedextemporaneously and advantageously contemporaneously withadministration of the preparation or shortly before administration ofthe preparation; for instance, shortly before or prior toadministration, the plasmid-adjuvant mixture is formed, advantageouslyso as to give enough time prior to administration for the mixture toform a complex, e.g. between about 10 and about 60 minutes prior toadministration, such as approximately 30 minutes prior toadministration.

When DOPE is present, the DMRIE:DOPE molar ratio is advantageously about95:about 5 to about 5:about 95, more advantageously about 1:about 1,e.g., 1:1.

The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be betweenabout 50:about 1 and about 1:about 10, such as about 10:about 1 andabout 1:about 5, and about 1:about 1 and about 1:about 2, e.g., 1:1 and1:2.

In another embodiment, pharmaceutically or veterinarily acceptablecarrier, excipient, adjuvant, or vehicle may be a water-in-oil emulsion.Examples of suitable water-in-oil emulsions include oil-basedwater-in-oil vaccinal emulsions which are stable and fluid at 4° C.containing: from 6 to 50 v/v % of an antigen-containing aqueous phase,preferably from 12 to 25 v/v %, from 50 to 94 v/v % of an oil phasecontaining in total or in part a non-metabolizable oil (e.g., mineraloil such as paraffin oil) and/or metabolizable oil (e.g., vegetable oil,or fatty acid, polyol or alcohol esters), from 0.2 to 20 p/v % ofsurfactants, preferably from 3 to 8 p/v %, the latter being in total orin part, or in a mixture either polyglycerol esters, said polyglycerolesters being preferably polyglycerol (poly)ricinoleates, orpolyoxyethylene ricin oils or else hydrogenated polyoxyethylene ricinoils. Examples of surfactants that may be used in a water-in-oilemulsion include ethoxylated sorbitan esters (e.g., polyoxyethylene (20)sorbitan monooleate (TWEEN 80®), available from AppliChem, Inc.,Cheshire, Conn.) and sorbitan esters (e.g., sorbitan monooleate (SPAN80®), available from Sigma Aldrich, St. Louis, Mo.). In addition, withrespect to a water-in-oil emulsion, see also U.S. Pat. No. 6,919,084,e.g., Example 8 thereof, incorporated herein by reference. In someembodiments, the antigen-containing aqueous phase comprises a salinesolution comprising one or more buffering agents. An example of asuitable buffering solution is phosphate buffered saline. In anadvantageous embodiment, the water-in-oil emulsion may be awater/oil/water (W/O/W) triple emulsion (U.S. Pat. No. 6,358,500).Examples of other suitable emulsions are described in U.S. Pat. No.7,371,395.

The immunological compositions and vaccines according to the inventionmay comprise or consist essentially of one or more adjuvants. Suitableadjuvants for use in the practice of the present invention are (1)polymers of acrylic or methacrylic acid, maleic anhydride and alkenylderivative polymers, (2) immunostimulating sequences (ISS), such asoligodeoxyribonucleotide sequences having one or more non-methylated CpGunits (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion,such as the SPT emulsion described on page 147 of “Vaccine Design, TheSubunit and Adjuvant Approach” published by M. Powell, M. Newman, PlenumPress 1995, and the emulsion MF59 described on page 183 of the samework, (4) cation lipids containing a quaternary ammonium salt, e.g., DDA(5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponinor (8) other adjuvants discussed in any document cited and incorporatedby reference into the instant application, or (9) any combinations ormixtures thereof.

The oil in water emulsion (3), which is especially appropriate for viralvectors, can be based on: light liquid paraffin oil (Europeanpharmacopoeia type), isoprenoid oil such as squalane, squalene, oilresulting from the oligomerization of alkenes, e.g. isobutene or decene,esters of acids or alcohols having a straight-chain alkyl group, such asvegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate),glycerol tri(caprylate/caprate) and propylene glycol dioleate, or estersof branched, fatty alcohols or acids, especially isostearic acid esters.

The oil is used in combination with emulsifiers to form an emulsion. Theemulsifiers may be nonionic surfactants, such as: esters of on the onehand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol,polyglycerol or propylene glycol and on the other hand oleic,isostearic, ricinoleic or hydroxystearic acids, said esters beingoptionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymerblocks, such as Pluronic, e.g., L121.

Among the type (1) adjuvant polymers, preference is given to polymers ofcrosslinked acrylic or methacrylic acid, especially crosslinked bypolyalkenyl ethers of sugars or polyalcohols. These compounds are knownunder the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). Oneskilled in the art can also refer to U.S. Pat. No. 2,909,462, whichprovides such acrylic polymers crosslinked by a polyhydroxyl compoundhaving at least three hydroxyl groups, preferably no more than eightsuch groups, the hydrogen atoms of at least three hydroxyl groups beingreplaced by unsaturated, aliphatic radicals having at least two carbonatoms. The preferred radicals are those containing 2 to 4 carbon atoms,e.g. vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals can also contain other substituents, such asmethyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA)are especially suitable. They are crosslinked by allyl saccharose or byallyl pentaerythritol. Among them, reference is made to Carbopol 974P,934P and 971P.

As to the maleic anhydride-alkenyl derivative copolymers, preference isgiven to EMA (Monsanto), which are straight-chain or crosslinkedethylene-maleic anhydride copolymers and they are, for example,crosslinked by divinyl ether. Reference is also made to J. Fields etal., 1960.

With regard to structure, the acrylic or methacrylic acid polymers andEMA are preferably formed by basic units having the following formula:

in which: R1 and R2, which can be the same or different, represent H orCH3; x=0 or 1, preferably x=1; y=1 or 2, with x+y=2.For EMA, x=0 and y=2 and for carbomers x=y=1.

These polymers are soluble in water or physiological salt solution (20g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda(NaOH), to provide the adjuvant solution in which the expressionvector(s) can be incorporated. The polymer concentration in the finalimmunological or vaccine composition can range between about 0.01 toabout 1.5% w/v, about 0.05 to about 1% w/v, and about 0.1 to about 0.4%w/v.

The cytokine or cytokines (5) can be in protein form in theimmunological or vaccine composition, or can be co-expressed in the hostwith the immunogen or immunogens or epitope(s) thereof. Preference isgiven to the co-expression of the cytokine or cytokines, either by thesame vector as that expressing the immunogen or immunogens or epitope(s)thereof, or by a separate vector thereof.

The invention comprehends preparing such combination compositions; forinstance by admixing the active components, advantageously together andwith a carrier, cytokine, and/or diluent.

Cytokines that may be used in the present invention include, but are notlimited to, granulocyte colony stimulating factor (G-CSF),granulocyte/macrophage colony stimulating factor (GM-CSF), interferon α(IFNα), interferon β (IFNβ), interferon γ, (IFNγ), interleukin-1α(IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-3(IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6(IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9(IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12(IL-12), tumor necrosis factor α (TNFα), tumor necrosis factor β (TNFβ),polyinosinic and polycytidylic acid, cytidine-phosphate-guanosineoligodeoxynucleotides (CpG ODN), and transforming growth factor β(TGFβ). It is understood that cytokines can be co-administered and/orsequentially administered with the immunological or vaccine compositionof the present invention. Thus, for instance, the vaccine of the instantinvention can also contain an exogenous nucleic acid molecule thatexpresses in vivo a suitable cytokine, e.g., a cytokine matched to thishost to be vaccinated or in which an immunological response is to beelicited (for instance, a bovine cytokine for preparations to beadministered to bovines).

In the case of immunological composition and/or vaccine based on abaculovirus/insect cell-expressed polypeptides, a dose may include,about 1 μg to about 2000 μg, about 50 μg to about 1000 μg, and fromabout 100 μg to about 500 μg of CPV antigen, epitope or immunogen. Thedose may include about 10² to about 10²⁰ VLPs, about 10³ to about 10²⁰,about 10⁴ to about 10²⁰. The dose volumes can be between about 0.1 andabout 10 ml, between about 0.2 and about 5 ml. In general, the skilledperson is aware of many dosing strategies, and will be able to optimizedosing without the exercise of non-routing work.

In an aspect, the invention provides a combination vaccine comprising avirus-like particle (VLP) component and a modified-live virus (MLV)component, wherein both the VLP and the MLV are directed against thesame pathogen or disease, and wherein the combination vaccine overcomesmaternally-derived antibodies (MDA).

In some embodiments, the combination vaccine provides protectiveimmunity with a single dose.

In some embodiments, the pathogen or disease is canine parvovirus (CPV).

In some embodiments, the pathogen or disease is foot-and-mouth diseasevirus (FMDV).

In some embodiments, the VLP component of the combination comprises atleast at least 10% CPV VLPs (w/w) as a function of total proteincontent.

In some embodiments, the VLP component comprises at least 20% CPV VLPs(w/w).

In some embodiments, the CPV VLP is expressed by a baculovirus vector ininsect cells.

In some embodiments, the CPV VLP comprises at least one CPV capsidprotein.

In some embodiments, the CPV VLP comprises a CPV polypeptide having thesequence as set forth in SEQ ID NO: 1, 3, 4, 6, 8, 9 or 10; or, the CPVVLP comprises a CPV polypeptide having at least 90% identity a sequenceas set forth in SEQ ID NO: 1, 3, 4, 6, 8, 9 or 10.

In some embodiments, the CPV VLP comprises a CPV polypeptide encoded bya polynucleotide having the sequence as set forth in SEQ ID NO: 2, 5 or7; or, the CPV VLP comprises a CPV polypeptide encoded by apolynucleotide having at least 90% identity to a sequence as set forthin SEQ ID NO: 2, 5 or 7.

In some embodiments, the combination vaccine is not adjuvanted andoptionally comprises a pharmaceutically or veterinarily acceptablecarrier, excipient, or vehicle.

In another aspect, the invention provides a plasmid useful for producingCPV VLP, comprising a polynucleotide encoding a CPV antigen having asequence as set forth in SEQ ID NO: 1, 3, 4, 6, or 8-10, or apolynucleotide sequence having at least 90% identity to a sequence asset forth in SEQ ID NO: 1, 3, 4, 6, or 8-10.

In some embodiments, the polynucleotide comprises or consists of thesequence as set forth in SEQ ID NO: 2, 5, 7, 11 or 12.

In some embodiments, the plasmid consists of the sequence as set forthin SEQ ID NO:11 or 12.

In some embodiments, the plasmid is stably transformed into an insectcell, which expresses CPV VLPs.

In another aspect, the invention provides a substantially purified CPVempty capsid or CPV VLP expressed in insect cells, wherein the CPV emptycapsid or VLP comprises a polypeptide having a sequence as set forth inSEQ ID NO: 1, 3, 4, 6, or 8-10; or, the CPV empty capsid or VLPcomprises a polypeptide having at least 90% identity to a sequence asset forth in SEQ ID NO: 1, 3, 4, 6, or 8-10.

In some embodiments, the CPV empty capsid or VLP consists of apolypeptide having a sequence as set forth in SEQ ID NO: 1, 3, 4, 6, or8-10.

In another aspect, the invention provides a method of eliciting animmune response in an animal against CPV comprising administering to theanimal the compositions, vaccines, combination vaccines and VLPSdisclosed herein.

In some embodiments, the immune response protects vaccinates againstsubsequent exposure to virulent CPV. The exposure may be natural orexperimental.

In some embodiments, the immune response is elicited in the vaccinatedanimals regardless of the presence in said animals of high levels ofmaternally-derived antibodies (MDA) against CPV. “High levels” has theordinary meaning, and generally refers to levels of MDA that impede theability of prior art vaccines to elicit a strong protective response inMDA-positive animals.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLES

Unless otherwise described, construction of DNA inserts, plasmids andrecombinant viral or baculovirus vectors was carried out using thestandard molecular biology techniques described by J. Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989).

Example 1 Construction and Expression of CPV Capsid Antigens inBaculovirus/Insect Cells System

Objective: Generate a pVL1393-based transfer plasmid encoding the CPVcapsid protein of CPV (canine parvovirus, strain Souriou serotype 2c)optimized for insect cells and generation of the recombinant baculovirusBacMEB072 in order to express virus like particles (VLP). The referencesequence was strain Souriou 2C (Merial), having the designation GenbankBAD34656/Swissprot P61826 (SEQ ID NO: 1).

The CPV capsid gene (1755 bp) encodes a 584 amino acid polypeptide(containing no signal peptide). The sequence encoding the CPV VP2protein (SEQ ID NO: 1) was cloned and the corresponding DNA sequence wascodon-optimized (SEQ ID NO: 5) for insect cells. Potential functionaldomains are shown in Table 1 below.

TABLE 1 Potential functional domains are the following (according toSwiss-prot annotation on P61826) Putative domains From to (or position)Length Signal sequence no mature chain VP2 1-584 584 N-glycosylation25-47-64-180-443-505- 517 N-glycosylation 490-494 sites

Generation of Plasmid pMEB072.

The CPV capsid optimized for insect expression (SEQ ID NO: 1) was clonedinto commercial plasmid pVL1393 (Pharmingen) using the XbaI and Bam HIsites of both the vector and insert to generate the expression plasmidpMEB072.

Generation of Recombinant Baculovirus BacMEB072.

The baculovirus vector used was AcNPV modified by a lethal deletion thatis only rescued through homologous recombination (BaculoGold DNA,Pharmingen). Plasmid pMEB072 was used to generate a recombinantbaculovirus, encoding CPV capsid gene strain Souriou serotype 2c undercontrol of polyhedrin promoter, by homologous recombination. Spodopterafrugiperda (Sf) 9 insect cells were co-transfected with plasmid pMEB072and Bsu36I triple-cut linearized AcNPV DNA, according to manufacturer'sprotocol (Baculogold, Pharmingen). Recombinant baculovirus fromco-transfection supernatant were plaque purified twice. Five clones wereamplified (passage 1) at 28° C. at a 25 cm² monolayer flask scale.Infected cells and supernatants were analysed for CPV capsid expressionby Dot Blot using monoclonals specific of CPV capsid antigen(CPV103B10A). Clone 1 showed a correct Dot Blot profile. This clone wasfurther amplified (passage 2) at 28° C. at a 50 mL scale in Erlenmeyer(suspension) at 105 rpm. A third passage (passage 3) at a 200 mL scalewas performed to obtain virus stock used for protein expression. Thisvirus stock was then titrated by plaque assay. Virus stock was obtainedusing SF900II media, supplemented with 2% of FCS. After titrationrecombinant baculovirus stock (Passage 3) was used for proteinproduction in serum free medium.

Expression Analysis of Baculovirus BacMEB072

TABLE 2 Size PM Signal N-glyco- Disulfide loca- Plasmid (AA) (kDa)peptide Tag sylation bridge tion pMEB062 545 59.2 no no 10 No secre-potential tion sites

Insect cells (Sf9) were infected with BacMEB072 at a Multiplicity OfInfection (MOI) of 1 pfu/ml. Insect cells were grown at 105 rpm inSf900II medium without FCS for 4 days at 28° C. Protein production wasanalyzed by submitting whole Sf9 lysates and culture supernatant toSDS-PAGE (4-20%, Invitrogen), followed by Dot Blot with monoclonalantibody (CPV103B10A). The solubility of the expressed proteins wasstudied by lysing the cellular pellets in lysis buffer (50 mM Tris-HclpH8, 500 mM NaCl+anti protease), sonication 3× (15 sec at 20% power, 30sec wait, 15 sec at 20% power; 5 min lysis on ice). Soluble proteinswere separated from the insoluble material by centrifugation (30 min at11,000 ref at 4° C.).

A band at expected size was expressed in the cell pellet of infectedcells lysed and clarified as observed by coomassie staining. The proteinaccumulated in cells, but was also detected in the soluble fractionafter lysis. The identity of VP2 protein was confirmed by Dot Blot usingspecific monoclonal antibody against CPV capsid (CPV103B10A).

The electronic microscopy (EM) analysis confirmed correct auto-assemblyof the capsid protein into VLPs, which had a diameter of 25-30 nm, and acorrect morphology of parvovirus-like virions (FIG. 4). The optimalconditions for VLP production were to use MOI=0.1 and to harvest at 5days post-infection. Although other conditions are envisioned, theseparticular conditions yielded a concentration of about 10¹¹ VLPs per ml.

Example 2—Production of BacMEB073 Containing Truncated CPV VP2

The objective was to generate a pVL1393-based transfer plasmid encodingthe truncated VP2 capsid protein of CPV (canine parvovirus, strainSouriou serotype 2c) optimized for mammals, and then to generate thecorresponding recombinant baculovirus BacMEB073, expressing the VLP. The9 N-terminal amino acids (−9AA) were deleted in an effort to enhance VP2expression without preventing VLP formation (Hurtado et al. Journal ofVirology, August 1996). In addition, three amino acids were added (M, Land K) in an effort to improve capsid structure and formation (Gilbertet al., Journal of Nanobiotechnology, 2006). Prior to this disclosure,it was unknown what effect the introduction of both the deletion and theinsertions would have on CPV VP2 expression and subsequent capsidformation.

The truncated VP2 gene (deletion of 9 amino acids in the N-terminalreplaced by additional MLK) and optimized for mammals (Geneart) wasfurther cloned into plasmid pVL1393 using the Bam HI and Xba I sites ofboth the vector and insert.

Baculovirus vector generation and protein expression. Baculovirusvector: AcNPV modified by a lethal deletion which is only rescuedthrough homologous recombination (BaculoGold DNA, Pharmingen).

Generation of Recombinant Baculovirus BacMEB073.

Plasmid pMEB073 was used to generate a recombinant baculovirus, encodingtruncated CPV capsid gene of strain Souriou serotype 2c under control ofpolyhedrin promoter, by homologous recombination. Spodoptera frugiperda(Sf) 9 insect cells were co-transfected with plasmid pMEB073 and Bsu36Itriple-cut linearized AcNPV DNA, according to manufacturer's protocol(Baculogold, Pharmingen). Recombinant baculovirus from co-transfectionsupernatant were plaque purified twice. Five clones were amplified(passage 1) at 28° C. at a 25 cm² monolayer flask scale. Infected cellsand supernatants were analysed for CPV capsid expression by Dot Blotusing monoclonals specific of CPV capsid antigen (CPV103B10A). Clone 1showed a correct Dot Blot profile. This clone was further amplified(passage 2) at 28° C. at a 50 mL scale in Erlenmeyer (suspension) at 105rpm. A third passage (passage 3) at a 200 mL scale was performed toobtain virus stock used for protein expression. This virus stock wasthen titrated by plaque assay. Virus stock was obtained using SF900 IImedia, supplemented with 2% of FCS.

After titration recombinant baculovirus stock (Passage 3) was used forprotein production in serum free medium.

Expression Analysis

TABLE 3 Expected recombinants N-Glyco- Sub- Size PM signal sylationDisulfide cellular Plasmid (AA) (kDa) peptide site bridge locationpMEB073 578 64.1 no 11, 19, 40, 50, no secreted 58, 66, 72, 79, 80, 511

Insect cells (Sf9) were infected with BacMEB073 at MOI=1 pfu/ml. Insectcells were grown at 105 rpm in Sf900 II medium without FCS during 4 daysat 28° C. Protein production was analyzed by submitting whole Sf9lysates and culture supernatant to SDS-PAGE (4-20%, Invitrogen) followedby Dot Blot with monoclonal antibody (CPV103B10A).

The solubility of the expressed proteins was studied by lysing thecellular pellets in lysis buffer (50 mM Tris-Hcl pH8, 500 mM NaCl+antiprotease), sonication 3× (15″ at 20% of potency, 30″ wait, 15″ at 20%potency, 5′ break on ice). Soluble proteins were separated from theinsoluble material by centrifugation (30′ min at 11000 ref at 4° C.).

Results and conclusion. A band at expected size of 64 kDa is expressedin the cell pellet of infected cells lysed and clarified as observed bycoomassie staining. The protein accumulated in cells, but was detectablein the soluble fraction after lysis. The identity of VP2 protein wasconfirmed by Dot Blot using a specific CPV capsid mAb (CPV103B10A).

EM analysis confirmed auto-assembly of the capsid protein into VLPs witha diameter of 25-30 nm, as well as a correct morphology ofparvovirus—like virions at a concentration of 10¹² VLPs per ml (FIG. 4).VLP production was optimal at MOI=0.1 with harvest 5 days postinfection, although other conditions may also yield high levels of VLP.

Example 3 Vaccination of Canines with Baculovirus Expressed CPV CapsidProtein

The primary objective of the study was to assess and compare the safetyand immunogenicity of 2 Virus Like Particles (VLPs) of the CPV2c strainSouriou in puppies (i.e. BacMEB072 and BacMEB073, described in Examples1 and 2). A secondary objective was to assess the adjuvant effects ofaluminium hydroxide/saponin or iscomatrix on the immunogenicity of theVLPs produced by BacMEB072 and BacMEB073. Eight week-old puppies werevaccinated and surveyed as indicated in Table 4.

TABLE 4 Experimental design for truncated CPV VP2 VLP v. Full length CPVVP2 MLV study Whole blood Antigen: VLP Clinical (sodium Group CPV2Adjuvant Vaccination monitoring Sera heparin) A BacME073 None D0 & D28D0, D0+ D−7, D0*, D35, D42 (n = 6) (truncated) by SC with 4-6 h, D1, D2D7, D28, B BacME073 Gel Al(OH)₃ 1 ml D28, D35, D42, (n = 6) (truncated)(1.7 mg) + containing D28+ 4-6 h, D70 Saponin (12 25 μg of D29, D30haemolytic active units) ingredient C BacME072 None (n = 6)(non-truncated) D BacME072 Gel Al(OH)₃ (n = 6) (non-truncated) (1.7mg) + Saponin (12 haemolytic units) E BacME072 Iscomatrix (n = 6)(non-truncated) (75 μg) F Control Control (n = 5) *before vaccination

In terms of safety, Al(OH)₃/Saponin induced no general reactions nor anyother signs except local heat and swellings. Mild swelling was observedfor 3 to 4 days after each vaccination in most of the dogs. Iscomatrixappears to be a very safe adjuvant. In terms of immunogenicity,vaccination of puppies with VLP CPV2 was able to induce both humoral andcellular immune response with no major differences between truncated orfull length VP2 associated in VLPs. Notably, the persistence of humoralimmune response was increased when VLPs were adjuvanted either withAl(OH)₃/Saponin or with Iscomatrix with no significant differencebetween the two adjuvants investigated. Moreover, Iscomatrix adjuvantincreased the magnitude of IFN gamma response not only compared to thenon-adjuvanted group but also to Al(OH)₃ and Saponin group.

Example 4 Vaccination of Canines with Baculovirus Expressed CPV VLPs orMLV CPV (Strains Souriou or Bari)

The objective of the study was to compare the immune response in puppiesvaccinated with CPV VLP, Souriou CPV MLV or Bari CPV MLV. The firstexperiment compared 1.52 log 10 TCID50/mL (IFI) Souriou; 1.0 log 10TCID50/mL (HA) Bari; and pMEB072 VLP (results in FIG. 8). The secondexperiment compared 2.21 log 10 TCID50/mL (IFI) Souriou; 2.0 log 10TCID50/mL (HA) Bari; and pMEB073-produced VLP in Al(OH)₃+saponinadjuvant (results in FIG. 9). Finally, the third experiment compared3.81 login TCID50/mL (IFI) Souriou; 3.0 log 10 TCID50/mL (HA) Bari; andpMEB072-produced VLP in Al(OH)₃+saponin adjuvant (results in FIG. 10).Taken together, these results showed that CPV VLP, made according to theinstant disclosure, provided a comparable immune response when comparedto exemplar CPV MLV. Importantly, the VLPs were able to induce a morerapid immune response when compared to both the low and intermediatedoses of either MLV strain.

Example 5 Vaccination of Canines with CPV MLV or Baculovirus-ExpressedCPV Capsid Protein+MLV CPV

The objective of the study was to evaluate the immunogenicity ofdifferent vaccine candidates administered at different doses andsubcutaneously in puppies with maternal antibodies. Prior to this study,it was not known whether CPV VLP could overcome maternal antibodies toinduce protective immunity in puppies.

TABLE 5 Experimental design for CPV VLP v. CPV MLV study Vaccineadministered Blood Sample Group SC on Day 0 (6 mL/puppy) Analysis A CPVMLV CPV2 MLV D0*, D7, D14, Anti-CPV (n = 9) 5.5 log10 D21, D28, Antibody(1 ml) Determination B CPV VLP + CPV VLP D34, D42, (Hemagglu- CPV MLVTarget dose: D56 tination (n = 10) 500 μg (1 ml) + Inhibition and/orCPV2 MLV ELISA) 5.5 log10 (1 ml)

As indicated in FIG. 11, FIG. 12 and Table 6, CPV antibody titers weresignificantly higher in the MLV+VLP group. These data indicate that theaddition of CPV VLP to the vaccine formulation was sufficient toovercome the maternal antibodies, a surprising and unexpected result.The inventors envision that VLP are capturing a significant proportionof circulating CPV MDA which in turn allows the VLP and CPV MLV toactively immunize puppies.

TABLE 6 Anti-CPV antibody titers (IHA) according to days postvaccination Group ID 0 7 14 21 MLV 2252826 10 <5 2560 320 MLV 2253001 10<5 1280 <5 MLV 2284928 80 <5 <5 <5 MLV 2284973 80 20 <5 20 MLV 228497580 20 <5 10 MLV 2285160 <5 <5 <5 5 MLV 2285360 160 40 <5 20 MLV 228536740 <5 <5 20 MLV 2285387 10 5 <5 <5 MLV + VLP 2252827 80 640 1280 320MLV + VLP 2252828 5 160 5120 2560 MLV + VLP 2284818 10 640 2560 2560MLV + VLP 2284843 10 640 2560 1280 MLV + VLP 2284861 20 1280 1280 1280MLV + VLP 2284935 40 640 1280 320 MLV + VLP 2284941 80 640 1280 640MLV + VLP 2284943 20 320 1280 1280 MLV + VLP 2284978 40 1280 1280 1280MLV + VLP 2285000 80 320 640 640 MLV: Modified Live Vaccine (PRIMODOG ®5.5 log10/ml) VLP: Virus Like Particle, 500 μg

Example 6 Vaccination of Canines with Baculovirus-Expressed CPV CapsidProtein, MLV CPV or Adenovirus-Vectored CDV

The objective of the study was to evaluate the immunogenicity of severaldoses of different vaccine candidates administered via different routes.The different candidates were CPV VLP, and recombinant adenovirusesexpressing either CPV or CDV (canine distemper) genes.

TABLE 7 Experimental design for CPV VLP v. Adeno CPV v. Adeno CDV studyVaccine administered at D0 and D28** Group Dose and Route Samples TakenAnalyses A VLP+_1 VLP CPV Blood vials: Determination Cellular (n = 6)High dose D0, D7, D14, of anti-CDV and/or B B VLP+_2 (500 μl stock D21,D28, D35, antibodies Memory (n = 6) solution/dose) D42, D56 and (ELISAand/or Cell SC (1 mL) D63 Hemagglutination Immune C VLP−** VLP CPVHeparin blood Inhibition) Response (n = 6) Low dose (50 μl stock vials:solution/dose) D7, D31, D35, SC (1 mL) D56 D VLP_Oral VLP CPV (n = 6) 2ml undiluted stock solution Oral E Ad5_CPV+ Adeno CPV (n = 6) vAD3032high dose target dose (8.64 log 10 TCID 50/mL*) SC (1 mL) F Ad5_CPVAdeno CPV (n = 6) vAD3032 low dose (target dose 7.64 log 10 TCID 50/mL*)SC (1 mL) G Ad5_CDV− Adeno CDV Determination (n = 6) vAD3031 of anti-CDVlow dose antibodies (target dose 7.34 log (Seroneutralization) 10 TCID50/mL*) SC (1 mL) H Ad5_CDV+ Adeno CDV (n = 6) vAD3031 high dose (targetdose 8.34 log 10 TCID 50/mL*) SC (1 ml)

As indicated in FIG. 13, Groups A and E induced in puppies protectivelevels of CPV antibody titers.

Example 7 Vaccination of Canines with Baculovirus-Expressed CPV CapsidProtein (VLP), VLP+RECOMBITEK® C4, RECOMBITEK® C4 Alone or NOBIVAC® 3

The objective of the study was to assess the antibody response followingthe administration of various experimental multivalent vaccineformulations containing conventional CPV-2 MLV or baculovirus-expressed,CPV-2c recombinant Virus Like Particle Vaccine (VLP) and onecompetitor/commercial vaccine in dogs with CPV MDAs.

Forty 6-7 week old maternal derived antibodies (MDAs) positive beagledogs from bitches previously immunized for CPV during pregnancy, wererandomly assigned to four treatment groups (n=10 dogs) using litter andantibody titer. Titrations from blood samples collected on Day −27 wereused for the randomization. All dogs were vaccinated twice, 21 daysapart with the assigned vaccine according to Table 8 below. Puppies haveCPV maternally derived Ab (MDA) at D0.

TABLE 8 Experimental design for CPV VLP v. RECOMBITEK ® C4 and NOBIWAC ®study Vaccination No. Groups at Day 0 and Day 21 Titers Dogs 1 CPV VLP273.5 μg total protein 10 Test Vaccine #4 (1 ml) 7.1 Log HA Titer *VLPsCPV-2c 2 CPV VLP 273.5 μg total protein 10 #4 (0.5 ml) *VLPs CPV-2c +7.1 Log HA Titer RECOMBITEK ® C4 CPV: 6.9 TCID 50/ml #1 (CDV-CAV-CPi,CDV: 7.1 TCID 50/ml CPV2)*** CPi: 5.8 TCID 50/ml (1 ml) CAV2: 6.0 TCID50/ml 3 RECOMBITEK ® C4 CPV: 6.9 TCID 50/ml 10 #1 (CDV-CAV-CPi, CDV: 7.1TCID 50/ml CPV2)*** CPi: 5.8 TCID 50/ml (1 ml) CAV2: 6.0 TCID 50/ml 4NOBIVAC ® 3 Unknown 10 (CDV-CAV2-CPV2) (1 ml) *Virus Like Particles ofcanine Parvovirus VP2: VLPs CPV-2c **Administered on the same side asthe concurrent vaccine approximately 3 cm away ***CDV-CAV-CPi-CPV2vaccine is referred as C4 HA: Hemagglutination in 0.5 ml

Blood was collected from all dogs on Days 0, 7, 15, 21, 28, 35 and 42and the sera were tested for CPV antibodies by the HAI and SerumNeutralization Antibodies (SNA) assays.

The CPV HAI titers were reported as the inverse of the highest dilutionpreventing hemagglutination and a value <20 was considered negative.

Except for 2 dogs in group 3 (C4) all dogs in each group tested positiveto CPV by HAI on Day 0, prior to vaccination. Seroconversion followingvaccination was defined as an increase in titer by 4 fold or more fromDay 0. Following the first vaccination, 6 out 10 dogs vaccinated withthe VLP-CPV2c (Group 1) and 6 out 10 dogs vaccinated with VLP-CPV2c-C4(Group 2) seroconverted by Day 7. There were no responders in groups 3and 4 (i.e. groups not containing VLPs) on Day 7. By Day 21, 5 out 10dogs from Group 2 (VLP-CPV2c-C4), 4 out 10 from Group 1 (VLP-CPV2c), 3out 10 from Group 4 (NOBIVAC3®) and 2 out 10 from Group 3 (C4)seroconverted (FIG. 15).

Following the second vaccination, by Day 35 (2 weeks post-vax)seroconversion was observed in all dogs from Groups 2 (VLP-CPV2c-C4) and4 (NOBIVAC3®) followed by 9 in Group 3 (C4) and 8 in Group 1(VLP-CPV2c). The VLP+C4 (Group 2) treatment induced the highestgeometric mean antibody titer throughout the study. See FIG. 17. Sevendays after V2, on Day 28, the GMT of this group was −7.5 times higherthan VLP alone (GMT=520), −9 times higher than C4 alone (GMT=422) and−28 times higher than the NOBIVAC3® group (GMT=139).

TABLE 9 IHA Titers by ID and Day for CPV VLP v. RECOMBITEK ® C4 andNOBIVAC ® study Day Group Vaccine Dog ID 0 7 15 21 28 35 42 1 VLP CPV-2cLCR-5 80 40 <20 <20 40 40 40 LGR-5 40 40 <20 <20 640 1280 1280 LRR-5 20160 160 160 10240 1280 1280 LVQ-5 20 80 80 80 2560 640 640 QQR-5 20 32040 20 160 160 160 RCR-5 40 320 80 20 640 640 640 RDR-5 20 160 320 6402560 1280 640 RGQ-5 20 320 320 320 1280 640 320 RLQ-5 80 80 40 <20 80160 320 RRR-5 80 40 20 <20 80 320 160 2 VLP CPV-2c + LDR-5 20 160 160 405120 1280 640 rDAPPI LHR-5 40 40 <20 <20 1280 640 640 LSQ-5 20 1280 1280640 20480 5120 2560 LWQ-5 40 160 5120 2560 20480 5120 5120 PKR-5 40 20<20 <20 1280 1280 1280 PLQ-5 40 80 1280 640 20480 5120 5120 QRR-5 20 1601280 1280 5120 2560 2560 RHQ-S 20 320 640 1280 5120 2560 1280 ROQ-5 4040 20 <20 320 640 640 RSQ-5 40 160 40 40 1280 640 320 3 rDAPPI LPR-5 20<20 160 160 1280 640 640 LQR-5 20 <20 160 160 640 640 320 LTQ-5 20 <20<20 <20 640 320 640 PIR-5 20 <20 <20 <20 160 320 640 POQ-5 <20 <20 <20<20 1280 640 1280 QSR-5 40 20 20 <20 160 320 160 RER-5 <20 <20 <20 <20640 320 640 RIQ-5 20 <20 <20 <20 1280 320 640 RPQ-5 80 40 20 20 <20 <20<20 RTQ-5 20 20 <20 <20 640 320 320 4 Nobivac LER-5 40 20 <20 <20 <20640 640 LFR-5 20 <20 <20 <20 640 1280 1280 LUQ-5 40 20 <20 <20 40 12801280 PJR-5 20 <20 <20 <20 <20 640 640 QTR-5 40 20 <20 <20 160 2560 1280RFR-5 20 <20 1280 640 1280 1280 1280 RJQ-5 20 <20 80 640 1280 2560 1280RKR-5 40 20 20 <20 40 1280 1280 RQR-5 40 20 <20 <20 160 320 320 RUQ-5 4020 20 640 160 320 640

TABLE 10 Summary Statistics for CPV VLP v. RECOMBITEK ® C4 and NOBIVAC ®study Geometric Arithmetic Day Group Vaccine N Mean Mean Median Min Max0 1 VLP CPV-2c 10 34.82 42.00 30 20.0 80.0 2 VLP CPV-2c + 10 30.31 32.0040 20.0 40.0 rDAPPI 3 rDAPPI 10 24.62 28.00 20 20.0 80.0 4 Nobivac 1030.31 32.00 40 20.0 40.0 7 1 VLP CPV-2c 10 113.14 156.00 120 40.0 320.02 VLP CPV-2c + 10 121.26 242.00 160 20.0 1280.0 rDAPPI 3 rDAPPI 10 21.4422.00 20 20.0 40.0 4 Nobivac 10 20.00 20.00 20 20.0 20.0 15 1 VLP CPV-2c10 64.98 110.00 60 20.0 320.0 2 VLP CPV-2c + 10 226.27 986.00 400 20.05120.0 rDAPPI 3 rDAPPI 10 30.31 48.00 20 20.0 160.0 4 Nobivac 10 34.82152.00 20 20.0 1280.0 21 1 VLP CPV-2c 10 52.78 132.00 20 20.0 640.0 2VLP CPV-2c + 10 171.48 654.00 340 20.0 2560.0 rDAPPI 3 rDAPPI 10 30.3148.00 20 20.0 160.0 4 Nobivac 10 56.57 206.00 20 20.0 640.0 28 1 VLPCPV-2c 10 519.84 1828.00 640 40.0 10240.0 2 VLP CPV-2c + 10 3880.238096.00 5120 320.0 20480.0 rDAPPI 3 rDAPPI 10 422.24 674.00 640 20.01280.0 4 Nobivac 10 139.29 380.00 160 20.0 1280.0 35 1 VLP CPV-2c 10422.24 644.00 640 40.0 1280.0 2 VLP CPV-2c + 10 1810.19 2496.00 1920640.0 5120.0 rDAPPI 3 rDAPPI 10 298.57 386.00 320 20.0 640.0 4 Nobivac10 970.06 1216.00 1280 320.0 2560.0 42 1 VLP CPV-2c 10 367.58 548.00 48040.0 1280.0 2 VLP CPV-2c + 10 1371.87 2016.00 1280 320.0 5120.0 rDAPPI 3rDAPPI 10 367.58 530.00 640 20.0 1280.0 4 Nobivac 10 905.10 992.00 1280320.0 1280.0 * The ‘<’ signs were removed from the values in order tocalculate the summary statistics.

Example 8 Vaccination of Canines with Baculovirus-Expressed CPV CapsidProtein (VLP), VLP+RECOMBITEK® C4, RECOMBITEK® C4 Alone or NOBIVAC® 3

The objective of this study was to assess the CPV antibody responsefollowing the administration of experimental Virus Like Particles madeof the VP2 capsid protein of canine Parvovirus type 2c (VLPs CPV-2c) atdifferent titers in combination with a CPV-2 MLV multivalent vaccineformulation and one commercial vaccine in dogs with CPV MDAs.

Fifty 6-7 week old maternal derived antibodies (MDAs) positive beagledogs from bitches previously immunized for CPV during pregnancy, wererandomly assigned to five treatment groups (n=10 dogs per group) usinglitter and antibody titer. Titrations from blood samples collected onDay-15 were used for the randomization.

Vaccines were prepared by rehydrating a lyophilized component with adiluent component. The Lyophilized serial used for the Test vaccines #1,#2, #3 and #5 was the same experimental 4-way (C4) vaccine. The titersfor each component were as follow: CPV:6.1 Log TCID50/ml (˜0.2 ml of 1×culture), CDV: 7.3 Log TCID50/ml, CPi: 5.5 Log TCID50/ml and CAV2:6.0Log TCID50/ml. All dogs were vaccinated twice, 21 days apart with theassigned vaccine according to Table 11 below.

TABLE 11 Experimental design for CPV VLP v. RECOMBITEK ® C4 andNOBIVAC ® study GROUP Vaccine (n = 10) Lyophilized component VaccineDiluent component 1 CDV-CAV2-CPi-CPV VLPs CPV-2c; lot #: 14Dec15 C4 +High Undiluted dose VLP- 7.0 Log aHA/ml CPV2c ~10 ml of 1X culture 2CDV-CAV2-CPi-CPV VLPs CPV-2c; lot #: 14Dec15 C4 + Mid Diluted 1:5 inwater dose VLP- 6.3 Log aHA/ml CPV2c ~2 ml of 1X culture 3CDV-CAV2-CPi-CPV VLPs CPV-2c; lot #: 14Dec15 C4 + Low Diluted 1:25 inwater dose VLP- 5.7 Log aHA/ml CPV2c ~0.4 ml of 1X culture 4 Nobivac ®Canine 3 Unknown Nobivac ® (CDV-CAV2-CPV2) 3 DAPv (1 ml) 5CDV-CAV2-CPi-CPV Water C4 No VLP- CPV2c

Blood was collected from all dogs on Days 0, 7, 15, 21, 28, 34 and 42and the sera were tested for CPV antibodies by the HAI assay.

The CPV HAI titers were reported as the inverse of the highest dilutionpreventing hemagglutination and a value <20 was considered negative. Alldogs in each group tested positive to CPV by HAI on Day 0, prior tovaccination (GMT average titers for all groups between 42.87 and 80).Seroconversion following vaccination was defined as an increase in titerby 4 fold or more from Day 0. Dogs seroconverting were categorized asresponders (FIG. 18). Seven days after the first vaccination, 5 out 10dogs vaccinated with the high dose VLP-CPV2c-C4 (Group 1) and 1 out 10dogs vaccinated with mid dose VLP-CPV2c-C4 (Group 2) seroconverted.There were no responders in groups 3, 4 and 5 (i.e. groups containinglow or no VLPs) on Day 7. Twenty-one days after the first vaccination,all dogs from Group 1 (high dose VLP-CPV2c-C4), 3 out 10 from Group 2(mid dose VLP-CPV2c-C4), 1 out 10 from Group 3 (low dose VLP-CPV2c-C4).There were no responders in groups 4 and 5 (i.e. groups not containingVLPs) (FIG. 18).

On Day 34, 13 days following the second vaccination, seroconversion wasobserved in all dogs from Groups 1 (high dose VLP-CPV2c-C4) and 4(NOBIVAC3®) followed by 9 in Group 2 (mid dose VLP-CPV2c-C4) 6 in Group3 (low dose VLP-CPV2c-C4) and 1 in Group 5 (C4 no VLP).

As shown in FIG. 19, the high dose VLP+C4 (Group 1) treatment inducedthe highest geometric mean antibody titer throughout the study. Thirteendays after V2, on Day 34, the GMT of this group was 4457.22, followed bythe mid dose VLP-CPV2c-C4 and the NOBIVAC3® groups; (GMT=1470.33), thelow dose VLP-CPV2c-C4 (GMT=298.57) and C4 alone (GMT=45.95).

TABLE 12 HI Results by Group, ID and Day for second CPV VLP v.RECOMBITEK ® C4 and NOBIVAC ® study Day Group *Vaccine ID 0 7 15 21 2834 42 1 Test Vaccine #1 PFE6 40 320 640 1280 5120 5120 2560 (rDAPP;C4) + VLPs PRD6 40 5120 5120 2560 5120 5120 2560 CPV-2c 7.1 Log HA QFC640 5120 5120 5120 10240 10240 5120 QOE6 20 160 2560 2560 10240 5120 5120RIE6 80 80 40 20 1280 2560 2560 RKD6 80 320 1280 640 10240 5120 2560SPF6 160 80 2560 1280 10240 10240 5120 TAF6 80 160 1280 640 10240 51205120 VAF6 80 160 80 80 640 1280 1280 VCE6 40 80 80 40 5120 2560 2560 GMT56.57 298.57 735.17 519.84 5120.00 4457.22 3151.73 2 Test Vaccine #2PWF6 40 160 320 160 2560 2560 1280 (rDAPP; C4) + VLPs QBF6 160 160 80 40640 640 320 CPV-2c 6.4 Log HA QLE6 40 80 1280 2560 10240 10240 5120 RJD680 80 320 160 2560 1280 1280 RWE6 80 80 20 <20 80 2560 1280 RXE6 40 8020 20 320 640 640 STE6 80 40 20 20 40 640 320 SUF6 40 40 40 20 160 51202560 TBF6 80 40 40 40 320 2560 1280 VDE6 80 40 40 20 40 160 160 GMT64.98 69.64 74.64 56.57 393.97 1470.33 905.10 3 Test Vaccine #3 QDE6 8040 20 20 40 80 80 (rDAPP; C4) + VLPs QED6 40 40 320 320 2560 2560 1280CPV-2c 5.7 Log HA QKE6 40 20 <20 <20 40 160 160 RGF6 40 80 40 20 40 1280640 RPD6 40 40 20 20 40 1280 640 RTF6 40 40 20 <20 80 1280 1280 SCE6 2020 <20 <20 1280 1280 1280 SQF6 80 40 40 20 40 160 160 UOE6 40 20 20 <2040 20 <20 UXF6 40 40 20 20 20 <20 <20 GMT 42.87 34.82 30.31 26.39 85.74298.57 242.51 4 Nobivac ® Canine PCE6 20 <20 <20 <20 40 640 640 3-DAPvQCF6 80 40 20 <20 <20 2560 640 RFF6 160 40 20 20 20 1280 640 RLD6 160160 20 <20 <20 2560 1280 ROD6 160 40 20 <20 <20 1280 1280 SDE6 40 40 20<20 40 1280 1280 SWE6 40 20 <20 <20 <20 640 320 SXE6 40 20 20 <20 <201280 1280 TCE6 160 40 <20 20 <20 2560 1280 UYF6 160 160 40 40 <20 25601280 GMT 80.00 42.87 21.44 21.44 22.97 1470.33 905.10 5 Test Vaccine #5PBE6 20 <20 <20 <20 80 1280 640 (rDAPP; C4) REF6 80 40 20 <20 <20 <20<20 RHE6 160 20 <20 <20 <20 <20 <20 RUE6 80 80 40 20 <20 <20 <20 SVF6160 <20 <20 <20 <20 <20 <20 TDE6 160 160 40 <20 <20 <20 <20 ULF6 20 20<20 <20 <20 320 320 UPE6 80 20 <20 <20 <20 80 320 UZF6 160 160 40 20 20<20 <20 VBF6 80 20 20 20 <20 <20 <20 GMT 80.00 37.32 24.62 20.00 22.9745.95 49.25 *For the purpose of Example 8, the below names will be usedin the following tables and charts. *C4 + High dose VLP-CPV2c = TestVaccine #1 *C4 + Mid dose VLP-CPV2c = Test Vaccine #2 *C4 + Low doseVLP-CPV2c = Test Vaccine #3 *C4 + No VLP-CPV2c = Test Vaccine #5

TABLE 13 Summary Statistics* by group for second CPV VLP v. RECOMBITEK ®C4 and NOBIVAC ® study Geometric Arithmetic Day Group Vaccine N MeanMean Median Min Max 0 1 C4 + High dose VLP-CPV2c 10 56.57 66.00 60 20.0160.0 2 C4 + Mid dose VLP-CPV2c 10 64.98 72.00 80 40.0 160.0 3 C4 + lowdose VLP-CPV2c 10 42.87 46.00 40 20.0 80.0 4 Nobivac ® Canine 3-DAPv 1080.00 102.00 120 20.0 160.0 5 C4 + No VLP-CPV2c 10 80.00 100.00 80 20.0160.0 7 1 C4 + High dose VLP-CPV2c 10 298.57 1160.00 160 80.0 5120.0 2C4 + Mid dose VLP-CPV2c 10 69.64 80.00 80 40.0 160.0 3 C4 + low doseVLP-CPV2c 10 34.82 38.00 40 20.0 80.0 4 Nobivac ® Canine 3-DAPv 10 42.8758.00 40 20.0 160.0 5 C4 + No VLP-CPV2c 10 37.32 56.00 20 20.0 160.0 151 C4 + High dose VLP-CPV2c 10 735.17 1876.00 1280 40.0 5120.0 2 C4 + Middose VLP-CPV2c 10 74.64 218.00 40 20.0 1280.0 3 C4 + low dose VLP-CPV2c10 30.31 54.00 20 20.0 320.0 4 Nobivac ® Canine 3-DAPv 10 21.44 22.00 2020.0 40.0 5 C4 + No VLP-CPV2c 10 24.62 26.00 20 20.0 40.0 21 1 C4 + Highdose VLP-CPV2c 10 519.84 1422.00 960 20.0 5120.0 2 C4 + Mid doseVLP-CPV2c 10 56.57 306.00 30 20.0 2560.0 3 C4 + low dose VLP-CPV2c 1026.39 50.00 20 20.0 320.0 4 Nobivac ® Canine 3-DAPv 10 21.44 22.00 2020.0 40.0 5 C4 + No VLP-CPV2c 10 20.00 20.00 20 20.0 20.0 28 1 C4 + Highdose VLP-CPV2c 10 5120.00 6848.00 7680 640.0 10240.0 2 C4 + Mid doseVLP-CPV2c 10 393.97 1696.00 320 40.0 10240.0 3 C4 + low dose VLP-CPV2c10 85.74 418.00 40 20.0 2560.0 4 Nobivac ® Canine 3-DAPv 10 22.97 24.0020 20.0 40.0 5 C4 + No VLP-CPV2c 10 22.97 26.00 20 20.0 80.0 34 1 C4 +High dose VLP-CPV2c 10 4457.22 5248.00 5120 1280.0 10240.0 2 C4 + Middose VLP-CPV2c 10 1470.33 2640.00 1920 160.0 10240.0 3 C4 + low doseVLP-CPV2c 10 298.57 812.00 720 20.0 2560.0 4 Nobivac ® Canine 3-DAPv 101470.33 1664.00 1280 640.0 2560.0 5 C4 + No VLP-CPV2c 10 45.95 182.00 2020.0 1280.0 42 1 C4 + High dose VLP-CPV2c 10 3151.73 3456.00 2560 1280.05120.0 2 C4 + Mid dose VLP-CPV2c 10 905.10 1424.00 1280 160.0 5120.0 3C4 + low dose VLP-CPV2c 10 242.51 556.00 400 20.0 1280.0 4 Nobivac ®Canine 3-DAPv 10 905.10 992.00 1280 320.0 1280.0 5 C4 + No VLP-CPV2c 1049.25 142.00 20 20.0 640.0

TABLE 14 Number of Responders (Serocoversion) by Group and Day; 1 = Yes,0 = No; Serocoversion is defined as a 4 fold or greater increase intiter from baseline. Day Group Vaccine ID 0 7 15 21 28 34 42 1 C4 + Highdose PFE6 0 1 1 1 1 1 1 VLP-CPV2c PRD6 0 1 1 1 1 1 1 QFC6 0 1 1 1 1 1 1QOE6 0 1 1 1 1 1 1 RIE6 0 0 0 0 1 1 1 RKD6 0 1 1 1 1 1 1 SPF6 0 0 1 1 11 1 TAF6 0 0 1 1 1 1 1 VAF6 0 0 0 0 1 1 1 VCE6 0 0 0 0 1 1 1 Total 0 5 77 10 10 10 2 C4 + Mid dose PWF6 0 1 1 1 1 1 1 VLP-CPV2c QBF6 0 0 0 0 1 11 QLE6 0 0 1 1 1 1 1 RJD6 0 0 1 1 1 1 1 RWE6 0 0 0 0 0 1 1 RXE6 0 0 0 01 1 1 STE6 0 0 0 0 0 1 1 SUF6 0 0 0 0 1 1 1 TBF6 0 0 0 0 1 1 1 VDE6 0 00 0 0 0 0 Total 0 1 3 3 7 9 9 3 C4 + low dose QDE6 0 0 0 0 0 0 0VLP-CPV2c QED6 0 0 1 1 1 1 1 QKE6 0 0 0 0 0 1 1 RGF6 0 0 0 0 0 1 1 RPD60 0 0 0 0 1 1 RTF6 0 0 0 0 0 1 1 SCE6 0 0 0 0 1 1 1 SQF6 0 0 0 0 0 0 0UOE6 0 0 0 0 0 0 0 UXF6 0 0 0 0 0 0 0 Total 0 0 1 1 2 6 6 4 Nobivac ®Canine PCE6 0 0 0 0 0 1 1 3-DAPv QCF6 0 0 0 0 0 1 1 RFF6 0 0 0 0 0 1 1RLD6 0 0 0 0 0 1 1 ROD6 0 0 0 0 0 1 1 SDE6 0 0 0 0 0 1 1 SWE6 0 0 0 0 01 1 SXE6 0 0 0 0 0 1 1 TCE6 0 0 0 0 0 1 1 UYF6 0 0 0 0 0 1 1 Total 0 0 00 0 10 10 5 C4 + No PBE6 0 0 0 0 1 1 1 VLP-CPV2c REF6 0 0 0 0 0 0 0 RHE60 0 0 0 0 0 0 RUE6 0 0 0 0 0 0 0 SVF6 0 0 0 0 0 0 0 TDE6 0 0 0 0 0 0 0ULF6 0 0 0 0 0 1 1 UPE6 0 0 0 0 0 0 1 UZF6 0 0 0 0 0 0 0 VBF6 0 0 0 0 00 0 Total 0 0 0 0 1 2 3

Accordingly, CPV VLPs promoted an earlier onset of immunity (OOI) thanMLV alone in MDA+ puppies, and CPV VLPs synergize with MLV C4 in MDA+puppies. Applicant submits that these results were unexpected andextremely favorable. Overcoming maternally-derived antibodies has longchallenged vaccine biologists, and these data indicate that the VLP+MLVapproach disclosed herein may be applied broadly to the problem ofproviding MDA+ offspring with protective immunity.

What we claim is:
 1. A combination vaccine comprising a canineparvovirus (CPV) virus-like particle (VLP) component and a CPVmodified-live virus (MLV) component, wherein both the VLP and the MLVare directed against the same pathogen or disease, wherein thecombination vaccine overcomes CPV-specific, maternally-derivedantibodies (MDA), wherein the pathogen is CPV; and wherein the CPV MLVand CPV VLP are present in amounts such that the CPV VLPs synergize theinduction of an immune response with the CPV MLV in MDA+ puppies whenadministered simultaneously.
 2. The combination vaccine of claim 1,which provides protective immunity with a single dose.
 3. Thecombination vaccine of claim 1, wherein the CPVMLV component is a CPV-2and the CPVVLP component is a CPV-2c.
 4. The combination vaccine ofclaim 1, further comprising a pharmaceutically or veterinarilyacceptable carrier.
 5. The combination vaccine of claim 3, wherein theVLP component of the combination comprises at least 10% CPV VLPs (w/w)as a function of total protein content.
 6. The combination vaccine ofclaim 5, wherein the VLP component comprises at least 20% CPV VLPs(w/w).
 7. The combination vaccine of claim 3, wherein the CPV VLP isexpressed by a baculovirus vector in insect cells.
 8. The combinationvaccine of claim 3, wherein the CPV VLP comprises at least one CPVcapsid protein.
 9. The combination vaccine of claim 3, wherein the CPVVLP comprises a CPV polypeptide having the sequence as set forth in SEQID NO: 1, 3, 4, 6, 8, 9 or 10; or, wherein the CPV VLP comprises a CPVpolypeptide having at least 90% identity a sequence as set forth in SEQID NO: 1, 3, 4, 6, 8, 9 or
 10. 10. The composition or vaccine of claim1, wherein the CPV VLP comprises a CPV polypeptide encoded by apolynucleotide having the sequence as set forth in SEQ ID NO: 2, 5 or 7;or, wherein the CPV VLP comprises a CPV polypeptide encoded by apolynucleotide having at least 90% identity to a sequence as set forthin SEQ ID NO: 2, 5 or
 7. 11. The combination vaccine of claim 1, whereinthe composition or vaccine is not adjuvanted and optionally comprises apharmaceutically or veterinarily acceptable carrier, excipient, orvehicle.
 12. A plasmid useful for producing CPV VLP, comprising apolynucleotide encoding a CPV antigen having a sequence as set forth inSEQ ID NO: 1, 3, 4, 6, or 8-10, or a polynucleotide sequence having atleast 90% identity to a sequence as set forth in SEQ ID NO: 1, 3, 4, 6,or 8-10.
 13. The plasmid of claim 12, wherein the polynucleotidecomprises or consists of the sequence as set forth in SEQ ID NO: 2, 5,7, 11 or
 12. 14. The plasmid of claim 12, wherein the plasmid consistsof the sequence as set forth in SEQ ID NO:11 or
 12. 15. The plasmid ofclaim 12, which is stably transformed into an insect cell, whichexpresses CPV VLPs.
 16. A substantially purified CPV empty capsid or CPVVLP expressed in insect cells, wherein the CPV empty capsid or VLPcomprises a polypeptide having a sequence as set forth in SEQ ID NO: 1,3, 4, 6, or 8-10; or, wherein the CPV empty capsid or VLP comprises apolypeptide having at least 90% identity to a sequence as set forth inSEQ ID NO: 1, 3, 4, 6, or 8-10.
 17. The purified CPV empty capsid or CPVVLP of claim 16, wherein the CPV empty capsid or VLP consists of apolypeptide having a sequence as set forth in SEQ ID NO: 1, 3, 4, 6, or8-10.
 18. A method of vaccinating an animal against CPV comprisingadministering to the animal the composition of any one of claims 1-7, orthe CPV empty capsids or VLPs of claim
 16. 19. The method of claim 18,wherein the immune response is protective, and wherein the protectiveimmune response protects vaccinated animals against subsequent exposureto virulent CPV.
 20. The method of claim 19, wherein the protectiveimmune response is elicited in the vaccinated animals, regardless of thepresence in said animals of high levels of maternally-derived antibodies(MDA) against CPV.